Chimeric antigen receptor with BCMA specificity and application thereof

Abstract

A B cell maturation antigen (BCMA) is expressed on malignant plasma cells. The present invention provides BCMA-specific chimeric antigen receptors and cells expressing such chimeric antigen receptors. In certain embodiments, engineered cells expressing the chimeric antigen receptors of the invention are capable of inhibiting the growth of BCMA-expressing tumors. The engineered cells of the invention are useful for the treatment of diseases and conditions in which an up-regulated or induced BCMA-targeted immune response is desirable and / or therapeutically beneficial. For example, engineered cells expressing the BCMA-specific chimeric antigen receptors of the invention can be used to treat a variety of cancers, including multiple myeloma.

Description

[0001] This application is a divisional application of Chinese patent application 201980048197.7 (PCT / US2019 / 042452), whose application date is July 18, 2019 and whose invention name is “BCMA-specific chimeric antigen receptor and its use”.

[0002] Reference to a sequence listing

[0003] This application incorporates by reference a sequence listing submitted in computer readable form as file 10455WO01-Sequence.txt, created on July 17, 2019, containing 66,907 bytes. Technical Field

[0004] The present invention relates to chimeric antigen receptors (CARs) specific for B cell maturation antigen (BCMA) and engineered cells comprising such CARs and methods of using the same. Background Art

[0005] B cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a type III transmembrane protein that lacks a signal peptide and contains a cysteine-rich extracellular domain. BCMA, together with closely related proteins, promotes B cell survival at different stages of development. BCMA is expressed exclusively in cells of the B cell lineage, specifically in the interfollicular zones of germinal centers, as well as in plasmablasts and differentiating 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 ubiquitously expressed at elevated 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 Macroglobulinemia, Burkitt’s lymphoma, and diffuse large B-cell lymphoma. Tai et al., Immunotherapy, 7(11): 1187-1199, 2015.

[0006] Adoptive immunotherapy involves the transfer of autologous antigen-specific T cells generated in vitro and is a promising strategy for the treatment of viral infections and cancer. T cells for adoptive immunotherapy can be generated by expansion of antigen-specific T cells or by redirection of genetically engineered T cells.

[0007] New specificities have been successfully generated in T cells through gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. Typically, the binding portion of a CAR consists of an antigen binding domain of a single-chain antibody (scFv), which includes light and heavy chain variable fragments of a monoclonal antibody connected by a flexible linker. The signaling domain of the first generation of CARs is derived from the cytoplasmic region of CD3zeta or the gamma chain of the Fc receptor. The first generation of CARs has been shown to successfully redirect the cytotoxicity of T cells. However, they cannot provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules as well as transmembrane and hinge domains have been added to form second and third generation CARs, resulting in some successful therapeutic trials in humans. For example, CAR redirection T cells specific for the B cell differentiation antigen CD19 have shown significant efficacy in the treatment of B cell malignancies, while TCR redirection T cells have shown benefits for patients with solid tumors. Stauss et al. describe strategies for modifying therapeutic CARs and TCRs for the treatment of cancer, such as enhancing antigen-specific effector functions and limiting the toxicity of engineered T cells (Current Opinion in Pharmacology 2015, 24: 113-118).

[0008] Engineered cells expressing chimeric antigen receptors targeting BCMA can be used in therapeutic settings where specific targeting and T cell-mediated killing of cells expressing BCMA is desired. Summary of the invention

[0009] In one aspect, the present invention provides a B cell maturation antigen (BCMA)-specific chimeric antigen receptor, which comprises, 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 co-stimulatory domain and a signaling domain.

[0010] In some cases, 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 cases, the chimeric antigen receptor further comprises a linker between the extracellular ligand binding domain (e.g., the scFv domain) and the hinge. In some cases, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-96. In some embodiments, the linker is (G4S) nLinker, wherein n is 1-10.

[0011] In some cases, the hinge, the transmembrane domain, or both are from a CD8 alpha polypeptide. In some cases, the costimulatory domain comprises a 4-1BB costimulatory domain. In some cases, the signaling domain comprises a CD3 zeta signaling domain. In some embodiments, the hinge comprises an amino acid sequence of SEQ ID NO: 97. In some embodiments, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 98. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD3 zeta signaling domain comprises an amino acid sequence of SEQ ID NO: 100.

[0012] In some cases, the LCVR comprises a complementarity determining region (CDR) of a 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 a 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 a 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 a 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.

[0013] 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 cases, 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.

[0014] In some embodiments, the scFv domain comprises a LCVR / HCVR amino acid sequence pair comprising an amino acid sequence of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50, or 74 / 66. In some cases, the LCVR and HCVR are connected by a linker, optionally via (G4S) n Connector connection, wherein n=1-3.

[0015] In some cases, the chimeric antigen receptor comprises an 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 an amino acid sequence of SEQ ID NO: 82. In some embodiments, the chimeric antigen receptor comprises an amino acid sequence of SEQ ID NO: 84. In some embodiments, the chimeric antigen receptor comprises an amino acid sequence of SEQ ID NO: 86. In some embodiments, the chimeric antigen receptor comprises an amino acid sequence of SEQ ID NO: 88. In some embodiments, the chimeric antigen receptor comprises an amino acid sequence of SEQ ID NO: 90.

[0016] In another aspect, the invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor as discussed above or herein. In some cases, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 81, 83, 85, 87 and 89.

[0017] In another aspect, the invention provides a vector comprising a nucleic acid molecule as discussed above or herein. In some cases, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0018] In another aspect, the invention provides a cell comprising a nucleic acid molecule or vector discussed above or herein. In some cases, the cell is a human T cell.

[0019] On the other hand, the invention provides an engineered cell comprising a chimeric antigen receptor discussed above or herein. In some cases, the engineered cell is an immune cell. In some cases, the immune cell is an immune effector cell. In some cases, 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.

[0020] In some cases, the engineered cells of the invention are used to treat BCMA-expressing cancers. In some cases, the BCMA-expressing cancer is multiple myeloma.

[0021] In another aspect, the present invention provides an engineered human T cell comprising a chimeric antigen receptor, which comprises, from N-terminus to C-terminus: (a) an extracellular ligand binding domain comprising an anti-BCMA single-chain variable fragment (scFv) domain, which includes 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 co-stimulatory domain and a CD3zeta signaling domain.

[0022] In some cases, the scFv domain comprises a LCVR / HCVR amino acid sequence pair comprising an amino acid sequence of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50, or 74 / 66. In some cases, the hinge comprises an amino acid sequence of SEQ ID NO: 97. In some cases, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 98. In some cases, the 4-1BB costimulatory domain comprises an amino acid sequence of SEQ ID NO: 99. In some cases, the CD3zeta signaling domain comprises an amino acid sequence of SEQ ID NO: 100.

[0023] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence of SEQ ID NO: 82. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence of SEQ ID NO: 84. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence of SEQ ID NO: 86. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence of SEQ ID NO: 88. In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence of SEQ ID NO: 90.

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising genetically modified human T cells and a pharmaceutically acceptable carrier, wherein the genetically modified human T cells comprise a chimeric antigen receptor as discussed above or herein. In some cases, the pharmaceutical composition is used to treat a BCMA-expressing cancer. In some embodiments, the BCMA-expressing cancer is multiple myeloma.

[0025] In another aspect, the invention provides an engineered cell as discussed above or herein. In some cases, the pharmaceutical composition is used to treat a BCMA-expressing cancer. In some embodiments, the BCMA-expressing cancer is multiple myeloma.

[0026] On the other hand, the present invention provides chimeric antigen receptors, nucleic acid molecules, vectors, cells or engineered cells discussed above or herein for use in the preparation of a drug for treating a cancer expressed by BCMA. In some cases, the cancer expressed by BCMA is multiple myeloma. In various embodiments, it is conceivable that the chimeric antigen receptors, nucleic acid molecules, vectors, cells or engineered cells discussed above or herein are used in any method discussed above or herein. For example, in some embodiments, the CAR or engineered cells discussed herein are used in drugs or for treating cancers discussed above or herein.

[0027] In another aspect, the present invention provides a method of enhancing T lymphocyte activity in a subject, comprising: introducing into the subject a T lymphocyte comprising a chimeric antigen receptor as discussed above or herein.

[0028] In another aspect, the present invention provides a method for treating a subject having cancer, comprising: introducing into the subject a therapeutically effective amount of T lymphocytes comprising a chimeric antigen receptor as discussed above or herein.

[0029] In another aspect, the present invention provides a method for stimulating a T cell-mediated immune response to 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 discussed above or herein.

[0030] 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 discussed above or herein.

[0031] In some embodiments of the methods discussed above, the subject is human. In some cases, the subject has multiple myeloma, B-lineage acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, leukemias and lymphomas, acute lymphocytic leukemia, Hodgkin's lymphoma, or childhood acute lymphocytic leukemia. In some embodiments, the subject has multiple myeloma.

[0032] On the other hand, the present invention provides a method for engineering a cell population to express a chimeric antigen receptor, wherein the method comprises: (a) providing an immune cell population; (b) introducing a nucleic acid molecule encoding a chimeric antigen receptor discussed above or herein into the immune cell; (c) culturing the immune cell under conditions expressing the nucleic acid molecule; and (d) isolating the immune cell expressing the chimeric antigen receptor on the cell surface. In some cases, the method further comprises: obtaining the immune cell population from a subject before introducing the nucleic acid molecule.

[0033] In another aspect, the present invention provides a method of treating a BCMA-expressing cancer in a subject, wherein the method comprises: (a) engineering a cell population according to the method discussed above; and (b) reintroducing the immune cell population expressing the chimeric antigen receptor into the subject. In some embodiments, the BCMA-expressing cancer is multiple myeloma.

[0034] Other embodiments will become apparent upon reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Exemplary nucleotide constructs for expressing chimeric antigen receptor (CAR) constructs are shown. Exemplary nucleotide constructs include anti-BCMA VL-linker-VH scFv, human CD8 hinge and transmembrane domains, 4-1BB costimulatory domains, CD3zeta signaling domains, and IRES:eGFP sequences for tracking CAR transduced cells. DETAILED DESCRIPTION

[0036] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments, rather than being restrictive, because the scope of the present invention is only limited by the appended claims. The features of any embodiment or embodiment can be combined with each other, and such a combination is clearly included in 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 list a range, which has a value representing the upper and lower ends of the range, and such a range is included in the scope of the present disclosure.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. As used herein, when used to refer to a specific numerical value listed, the term "about" means that the numerical value may differ from the referenced value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0038] Preferred methods and materials are now described, however any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0039] definition

[0040] As used herein, the expression "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 plasma cells that produce immunoglobulins. As used herein, "BCMA" refers to human BCMA protein, unless designated as from non-human species (e.g., "mouse BCMA", "monkey BCMA", etc.). Human BCMA protein has the amino acid sequence shown in SEQ ID NO:101.

[0041] As used herein, “antibodies that bind to BCMA” or “anti-BCMA antibodies” include antibodies and antigen-binding fragments thereof that specifically recognize BCMA.

[0042] 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 predetermined 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 constructs discussed in Example 2 include scFvs having variable regions derived from specific anti-BCMA antibodies. These anti-BCMA antibodies are the "corresponding antibodies" for the respective chimeric antigen receptors.

[0043] As used herein, the term "antibody" refers to any antigen binding molecule or molecular complex that includes at least one complementary determining region (CDR) that specifically binds or interacts with a specific antigen (e.g., BCMA). The term "antibody" includes immunoglobulin molecules, which include four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules that include four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region consists of three domains C H 1. C H 2 and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain (C L 1) You can set V H Area and V L The V region is further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L It includes three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-BCMA antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0044] As used herein, the term "antibody" also includes the antigen binding fragment of a complete antibody molecule. As used herein, the term "antigen binding portion thereof", "antigen binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein, which specifically binds to an antigen to form a complex. The antigen binding fragment of an antibody can be derived from a complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or involving manipulation and expression of DNA encoding variable and optionally constant domains of an antibody. Such DNA is known and / or is easy to obtain from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or by molecular biological techniques, for example, one or more variable and / or constant domains are arranged into a suitable configuration, or codons are introduced, cysteine ​​residues are produced, modified, added or deleted amino acids, etc.

[0045] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units, which consist of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variant IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0046] The antigen-binding fragment of an antibody generally includes at least one variable domain. The variable domain can have any size or amino acid composition and generally includes at least one CDR adjacent to or within one or more framework sequences. L Domain associated V H In the antigen-binding fragment of the domain, V H Domain and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or V L domain.

[0047] In certain embodiments, an antigen-binding fragment of an antibody may contain 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 within an antigen-binding fragment of an antibody of the invention include: (i) 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 -CH 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -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; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable and constant regions, including any of the exemplary configurations listed above, the variable domains and constant domains may be directly linked to each other or may be linked through a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the present invention may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, which are non-covalently associated with each other and / or with one or more monomer V H or V L The domains are non-covalently associated (e.g., via one or more disulfide bonds).

[0048] In certain embodiments, the anti-BCMA antibody is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), for example, in the CDR region, particularly in the CDR3 region. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences.

[0049] In some embodiments, the antibody can be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as: antibodies expressed using recombinant expression vectors transfected into host cells (described further below); antibodies isolated from recombinant, combinatorial human antibody libraries (described further 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, produced or isolated by any other means, including splicing 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 undergo in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, undergo in vivo somatic mutagenesis), and thus the V H Area and V L The amino acid sequence of the region is as follows: Although derived from human germline V H Sequence and V L sequences that are related to but may not naturally occur in vivo in the human antibody germline repertoire.

[0050] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule includes a stable four-chain construct of about 150-160 kDa, in which the dimer is held together by an interchain heavy chain disulfide bond. In the second form, the dimer is not connected by an interchain disulfide bond and forms a molecule of about 75-80 kDa, which is composed of covalently coupled light and heavy chains (half antibodies). Even after affinity purification, these forms are extremely difficult to separate.

[0051] The frequency of the second form in various intact IgG isotypes is based on, 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 the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed with the human IgG1 hinge. The present invention encompasses substitutions in the hinge, C H Zone 2 or C H Region 3: Antibodies with one or more mutations, which may be desirable, for example, in production to increase the yield of a desired antibody form.

[0052] The antibody can be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified and 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 the antibody naturally exists or is naturally produced, is an "isolated antibody" for purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or separation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular material and / or chemicals.

[0053] The anti-BCMA antibodies disclosed herein may include 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, as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof, which are derived from any amino acid sequence disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to one or more corresponding residues of the germline sequence from which the antibodies were derived, or mutated to one or more corresponding residues of another human germline sequence, or mutated to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can easily produce many antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LIn other embodiments, all frameworks and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence of the antibody derived. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only the residue after the mutation found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only the residue after the mutation found in CDR1, CDR2 or CDR3. In other embodiments, one or more mutations in the framework and / or one or more CDR residues are one or more corresponding residues of different germline sequences (that is, germline sequences different from the germline sequence of the antibody initially derived). In addition, the antibody of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR districts, for example, wherein some individual residues are mutated into the corresponding residues of specific germline sequences, and some other residues different from the original germline sequence can maintain or mutate into the corresponding residues of different germline sequences. 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 antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0054] The anti-BCMA antibodies may include variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the anti-BCMA antibodies may have HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequences set forth herein.

[0055] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule called a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope can include a portion of a sugar, phosphoryl, or sulfonyl group on the antigen.

[0056] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof means a nucleotide sequence identity of at least about 95%, more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence that is identical or substantially similar to a polypeptide encoded by the reference nucleic acid molecule.

[0057] When applied to these polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned using the default gap weights as by the programs GAP or BESTFIT. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is substituted by another amino acid residue having a similar side chain (R group) with chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups 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 substitutions 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, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0058] Sequence similarity of polypeptide is usually measured using sequence analysis software, also referred to as sequence identity. Protein analysis software uses measurements assigned to various substitutions, deletions and other modifications, including the similarity of conservative amino acid substitutions to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, and the program can be used together with default parameters to determine closely related polypeptides, such as sequence homology or sequence identity between homologous polypeptides from different organism species or between wild-type protein and its mutant protein. See, for example, GCG Version 6.1. FASTA (the program in GCG Version 6.1) can also be used to compare polypeptide sequences using default or recommended parameters. FASTA (for example, FASTA2 and FASTA3) provides alignment and sequence identity percentage (Pearson (2000), supra) of the best overlapping region between the query sequence and the search sequence. When comparing the sequence of the present invention with a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. 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.

[0059] 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 produced by polymerase chain reaction (PCR); and fragments produced by ligation, cleavage, endonuclease action and 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 (for example, enantiomeric forms of naturally occurring nucleotides), or a combination of the two. Modified nucleotides can have changes in sugar moieties and / or pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyls, amines and azido groups, or sugars can be functionalized as ethers or esters. In addition, the entire sugar moiety can be replaced by spatially 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 substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be single-stranded or double-stranded.

[0060] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, such as an antibody-based specificity for a desired antigen (e.g., a tumor antigen, such as BCMA), with a T cell receptor activating intracellular domain to produce a chimeric protein that exhibits specific anti-target cell immune activity. Typically, a CAR consists of an extracellular single-chain antibody binding domain (scFv) fused to the intracellular signaling domain of the Zeta chain of the T cell antigen receptor complex, and has the ability to specifically redirect antigen recognition based on a monoclonal antibody when expressed in T cells.

[0061] As used herein, the term "vector" includes but is not limited to viral vectors, plasmids, RNA vectors or linear or circular DNA or RNA molecules, which can be composed of chromosomes, non-chromosomes, semi-synthetic or synthetic nucleic acids. In some cases, a vector is a vector (additional vector) capable of autonomous replication and / or a vector (expression vector) capable of expressing a nucleic acid linked thereto. A large number of suitable vectors are known to those skilled in the art and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses)), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses) and double-stranded DNA viruses (including adenoviruses, herpes viruses (e.g., herpes simplex viruses type 1 and type 2, Epstein-Barr viruses, cytomegaloviruses) and poxviruses (e.g., vaccinia, fowlpox and canarypox)). For example, other viruses include Norwalk virus, Togavirus, Flavivirus, Reovirus, Pasteurvirus, Hepadnavirus and Hepatitis virus. Examples of retroviruses include: avian leukosis sarcoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV group and lentivirus.

[0062] "Costimulatory domain" or "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand to mediate a costimulatory response (such as but not limited to proliferation) of the cell. Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors. Examples of costimulatory molecules include 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 a ligand specifically bound to CD83, etc. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response.

[0063] "Co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses (including but not limited to proliferation activation, differentiation, etc.) in addition to providing a primary signal (e.g., by binding a TCR / CD3 complex to an MHC molecule loaded with a peptide). Co-stimulatory ligands may include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3.

[0064] A "co-stimulatory signal" refers to a signal that, in conjunction with a primary signal (such as TCR / CD3 ligation), results in T cell proliferation and / or up- or down-regulation of key molecules.

[0065] As used herein, the term "extracellular ligand binding domain" refers to an oligonucleotide or polypeptide that is capable of binding a ligand (e.g., a cell surface molecule). For example, an extracellular ligand binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state (e.g., cancer). Examples of cell surface markers that can act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0066] 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 suffering from cancer (eg, multiple myeloma).

[0067] As used herein, the "signal transduction domain" or "signaling domain" of CAR is responsible for intracellular signaling after the extracellular ligand binding domain binds to the target, thereby causing the activation and immune response of immune cells. In other words, the signal transduction domain is responsible for the activation of at least one normal effector function of the immune cells expressing CAR therein. For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines. Therefore, the term "signal transduction domain" refers to a part of a protein that transduces effector function signals and guides cells to perform specialized functions. Examples of signal transduction domains for CAR can be cytoplasmic sequences of T cell receptors and co-receptors (which work together to initiate signal transduction after antigen receptor engagement), as well as any derivatives or variants of these sequences and any synthetic sequences with the same function. In some cases, the signal transduction domain includes two different types of cytoplasmic signaling sequences: one type of cytoplasmic signaling sequence initiates antigen-related primary activation, while another type of cytoplasmic signaling sequence acts in a non-antigen-related manner to provide secondary or costimulatory signals. The primary cytoplasmic signaling sequence can include a signaling motif, which is referred to as an activation motif based on immunoreceptor tyrosine of ITAM. ITAM is a well-defined signaling motif present in the cytoplasmic tail of various receptors, which acts as a binding site for syk / zap70 class tyrosine kinases. Exemplary ITAMs include those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, the signal transduction domain of CAR may include CD3zeta signaling domain (SEQ ID NO: 100).

[0068] Chimeric Antigen Receptor (CAR)

[0069] Chimeric antigen receptors (CARs) redirect T cells specifically to antibody-recognized antigens expressed on the surface of cells (e.g., cancer cells), whereas T cell receptors (TCRs) expand the range of targets to include intracellular antigens (e.g., tumor antigens).

[0070] One aspect of the present invention includes a chimeric antigen receptor (CAR) that is specific for B cell maturation antigen (BCMA) expressed on the surface of malignant plasma cells. In one embodiment of the present invention, the CAR described herein includes an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (e.g., a signaling domain derived from CD3zeta or FcRgamma) and / or one or more costimulatory signaling domains derived from costimulatory molecules, such as but not limited to 4-1BB. In one embodiment, CAR includes a hinge or spacer between the extracellular binding domain and the transmembrane domain, such as a CD8alpha hinge.

[0071] The binding domain or extracellular domain of CAR enables CAR to have the ability to bind to the target target antigen. The binding domain (e.g., ligand binding domain or antigen binding domain) can be any protein, polypeptide, oligopeptide or peptide with 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 of the target biomolecule. For example, and as further described herein, the binding domain can be the light chain and heavy chain variable region of an antibody, or the light chain and heavy chain variable region can be single-chain and connected together in any orientation (e.g., VL-VH or VH-VL). It is known to be used to identify the various assays for the binding domains specifically bound to a specific target disclosed herein, including Western blotting, ELISA, flow cytometry or surface plasmon resonance analysis (e.g., using BIACORE analysis). The target can be an antigen required clinically, and it is expected to trigger an effector immune response against the antigen, triggering killing of the tumor. In one embodiment, the target antigen of the binding domain of the chimeric antigen receptor is the BCMA protein on the surface of tumor cells (particularly tumor cells of the B cell lineage, such as multiple myeloma cells).

[0072] Exemplary ligand binding domains include antigen binding proteins, such as antigen binding fragments of antibodies, such as scFv, scTCR, the extracellular domain of receptors, cell surface molecules / ligands of receptors or their receptor binding domains and tumor binding proteins. In certain embodiments, the antigen binding domains included in the CAR of the present invention can be variable regions (Fv), CDR, Fab, scFv, VH, VL, domain antibody variants (dAb), camelid antibodies (VHH), fibronectin 3 domain variants, ankyrin repeat variants and other antigen-specific binding domains derived from other protein scaffolds.

[0073] In one embodiment, the binding domain of CAR is an anti-BCMA single-chain antibody (scFv), and may be a murine, human or humanized scFv. Single-chain antibodies can be cloned from the V region genes of hybridomas specific for the desired target. For example, Orlandi et al., PNAS, 1989; 86: 3833-3837 have been described for cloning variable region heavy chains (VH) and variable region light chains (VL). Therefore, in certain embodiments, the binding domain includes an antibody-derived binding domain, but may be a non-antibody-derived binding domain. The antibody-derived binding domain may 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.

[0074] In certain embodiments, the CAR of the present invention may include a joint between each domain added for the appropriate spacing and conformation of the molecule. For example, in one embodiment, there may be a joint between the binding domain VH or VL, and its length may be between 1-10 amino acids. In other embodiments, the length of the joint between any domains of the chimeric antigen receptor may be between 1-20 amino acids or 20 amino acids long. In this regard, the joint 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 a further embodiment, the joint may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. The range including the numbers described herein is also included herein, for example, a joint with a length of 10-30 amino acids.

[0075] In some embodiments, the joint suitable for CAR described herein is a flexible joint. Suitable joints can be easily selected, and it can have any suitable different lengths, for example, from 1 amino acid (for example, Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids or 7 amino acids to 8 amino acids, and can be 1,2,3,4,5,6 or 7 amino acids.

[0076] Exemplary flexible linkers include glycine polymers (G) n, glycine-serine polymers (wherein n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively structureless and can therefore be used as neutral tethers between domains of fusion proteins (e.g., CAR described herein). Glycine accesses a space that is significantly more phi-psi than alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those of ordinary skill will recognize that the design of CAR may include all or part of a flexible linker, such that the linker may include a flexible linker and one or more portions that impart a less flexible structure to provide a desired CAR structure. Specific linkers include (G4S) n Linkers, wherein n=1-3, as shown in SEQ ID NOs:95-97, and the linker shown in SEQ ID NO:96.

[0077] There may be a "spacer" or "hinge" after the binding domain of CAR, which refers to a region where the antigen binding domain is moved away from the effector cell surface to achieve appropriate cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge region in CAR is generally between the transmembrane (TM) and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, and may be a wild-type immunoglobulin hinge region or a changed wild-type immunoglobulin hinge region. Other exemplary hinge regions used in CAR described herein include hinge regions derived from the extracellular region of type 1 membrane proteins (such as CD8alpha, CD4, CD28 and CD7), which may be wild-type hinge regions from these molecules or may be changed. In one embodiment, the hinge region includes a CD8alpha hinge (SEQ ID NO: 97).

[0078] The "transmembrane" region or domain is a part of CAR that anchors the extracellular binding portion to the plasma membrane of immune effector cells and promotes the binding of the binding domain to the target antigen. The transmembrane domain can be a CD3zeta transmembrane domain, but other transmembrane domains that can be used include those obtained from CD8alpha, 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 includes the amino acid sequence of SEQ ID NO:98. In certain embodiments, the transmembrane domain is synthetic, in which case it will mainly include hydrophobic residues, such as leucine and valine.

[0079] "Intracellular signaling domain" or "signaling domain" refers to a part of a chimeric antigen receptor protein that is involved in transducing information of an effective CAR bound to a target antigen to the interior of an immune effector cell to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to target cells bound to CAR, or other cellular responses caused by antigens bound to the extracellular CAR domain. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or an activity that helps or includes cytokine secretion. Therefore, the terms "intracellular signaling domain" or "signaling domain" used interchangeably herein refer to portions of proteins that transduce effector function signals and guide cells to perform specialized functions. Although the entire intracellular signaling domain can generally be used, in many cases, the entire domain does not have to be used. In terms of using a truncated portion of an intracellular signaling domain, as long as it transduces an effector function signal, the truncated portion can be used instead of the entire domain. The term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal. The intracellular signaling domain is also referred to as a "signaling domain" and is typically derived from a portion of the human CD3 or FcRy chain.

[0080] It is known that the signal generated only by the T cell receptor is not enough to fully activate T cells, and secondary or costimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those sequences (primary cytoplasmic signaling sequences) that initiate antigen-related primary activation by the T cell receptor and those sequences (secondary cytoplasmic signaling sequences) that act in a non-antigen-related manner to provide secondary or costimulatory signals. The cytoplasmic signaling sequence that acts in a costimulatory manner can include a signaling motif, which is referred to as an activation motif or ITAM based on immunoreceptor tyrosine.

[0081] Examples of primary cytoplasmic signaling sequences containing ITAMs that are particularly used in the present invention include those derived from TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a specific embodiment, the intracellular signaling domain of the anti-BCMA CAR described herein is derived from CD3zeta. In some embodiments, the signaling domain includes the amino acid sequence of SEQ ID NO: 100.

[0082] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the part of the intracellular domain of the CAR including a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors, which provide a second signal required for the effective activation and function of T lymphocytes after binding to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and ligands specifically binding to CD83. Therefore, although the present disclosure provides exemplary costimulatory domains derived from CD3zeta and 4-1BB, other costimulatory domains are also considered for use with CAR described herein. Including one or more costimulatory signaling domains can enhance the efficacy and amplification of T cells expressing CAR receptors. Intracellular signaling and costimulatory signaling domains can be linked in series to the carboxyl terminus of the transmembrane domain in any order. In some embodiments, the costimulatory domain comprises the amino acid sequence of SEQ ID NO:99.

[0083] Although scFv-based CARs engineered to contain signaling domains from CD3 or FcRgamma have been shown to provide potent signals for T cell activation and effector function, they are insufficient to elicit signals that promote T cell survival and expansion in the absence of accompanying co-stimulatory signals. Other CARs that contain a binding domain, a hinge, a transmembrane and a signaling domain derived from CD3zeta or FcRgamma, and one or more co-stimulatory signaling domains (e.g., intracellular co-stimulatory domains derived from CD28, CD137, CD134, and CD278) can more effectively direct T cells expressing the CAR to have anti-tumor activity in vitro and in animal models and cancer patients, as well as increased cytokine secretion, lytic activity, survival, and proliferation (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).

[0084] In various embodiments, the BCMA CAR of the present invention includes: (a) anti-BCMA scFv as a binding domain (e.g., a scFv having a binding region (e.g., CDR or variable domain) from any one or more BCMA antibodies identified in Table 1), (b) a hinge region derived from human CD8alpha, (c) a human CD8alpha transmembrane domain, and (d) a human T cell receptor CD3zeta chain (CD3) intracellular signaling domain, and optionally one or more co-stimulatory signaling domains, such as 4-1BB. In one embodiment, the different protein domains are arranged in the following order from amino to carboxyl terminus: binding domain, hinge region, and transmembrane domain. The intracellular signaling domain and the optional co-stimulatory signaling domain are linked in series to the transmembrane carboxyl terminus in any order to form a single-chain chimeric polypeptide. In one embodiment, the nucleic acid construct encoding BCMA CAR is a chimeric nucleic acid molecule including a nucleic acid molecule, which includes different coding sequences, such as (5' to 3') human anti-BCMA scFv, human CD8alpha hinge, human CD8alpha transmembrane domain and CD3zeta intracellular signaling domain coding sequence. In another embodiment, the nucleic acid construct encoding BCMA CAR is a chimeric nucleic acid molecule including a nucleic acid molecule, which includes different coding sequences, such as (5' to 3') human anti-BCMA scFv, human CD8alpha hinge, human CD8alpha transmembrane domain, 4-1BB costimulatory domain and CD3zeta costimulatory domain coding sequence.

[0085] 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 so as to induce expression of the protein and / or cloning of the polynucleotide. Such a vector may also be referred to as an "expression vector". The isolated polynucleotide may be inserted into a vector using any suitable method known in the art, such as, but not limited to, the vector may be digested with a suitable restriction enzyme and then connected to an isolated polynucleotide having a matching restriction end. The expression vector has the ability to incorporate and express a heterologous or modified nucleic acid sequence encoding 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. The expression vector may include a variety of control sequences, which refer to nucleic acid sequences necessary for transcription and possible translation of an operatively connected coding sequence in a particular host organism. In addition to control sequences for transcription and translation, vectors and expression vectors may also include nucleic acid sequences having other functions, and are discussed below. The expression vector may include other elements, for example, the expression vector may have two replication systems so that it can be maintained in two organisms, such as expressed in human cells, and cloned and amplified in a prokaryotic host.

[0086] The expression vector may have necessary 5' upstream and 3' downstream regulatory elements, such as promoter sequences, such as CMV, PGK and EF1alpha promoters, ribosome recognition and binding TATA box, and 3'UTR AAUAAA transcription termination sequence, to carry out efficient gene transcription and translation in their respective host cells. Other suitable promoters include constitutive promoters of the 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. Human gene promoters may also be used, including but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. In certain embodiments, inducible promoters are also considered to be part of the vector expressing the 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 metallothioneine promoters, glucocorticoid promoters, progesterone promoters or tetracycline promoters.

[0087] The expression vector may have other sequences incorporated into the expressed CAR, such as 6x-histidine, c-Myc and FLAG tags. Therefore, the expression vector may be engineered to include 5' and 3' non-translated regulatory sequences, which may sometimes serve as such enhancer sequences, promoter regions and / or terminator sequences: which may promote or enhance the effective transcription of the target nucleic acid carried on the expression vector. The expression vector may also be modified for replication and / or expression function (e.g., transcription and translation) in a specific cell type, cell position or tissue type. The expression vector may include a selection marker for maintaining the vector in a host or recipient cell.

[0088] In various embodiments, the vector is a plasmid, an autonomously replicating sequence, and a transposable element. Other exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., lambda phages or M13 phages), and animal viruses. Examples of animal virus classes that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (e.g., SV40). Examples of expression vectors are Lenti-X for expression in mammalian cells. TMBicistronic expression system (Neo) vector (Clontrch), pClneo vector (Promega); pLenti4 / V5-DEST.TM., pLenti6 / V5-DEST.TM. and pLenti6.2N5-GW / lacZ (Invitrogen), for lentiviral-mediated gene transfer and expression in mammalian cells. The coding sequence of CAR disclosed herein can be connected to such expression vectors to express chimeric proteins in mammalian cells.

[0089] In certain embodiments, a nucleic acid encoding the CAR of the present invention is provided in a viral vector. A viral vector may be a vector derived from a retrovirus, a lentivirus or a foamy virus. As used herein, the term "viral vector" refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. A viral vector may include coding sequences of various chimeric proteins described herein of non-essential viral genes. Vectors and / or particles may be used for the purpose of transferring DNA, RNA or other nucleic acids to cells in vitro or in vivo. Various forms of viral vectors are known in the art.

[0090] In certain embodiments, the viral vector comprising the coding sequence of the CAR described herein is a retroviral vector or a lentiviral vector. The term "retroviral vector" refers to a vector containing structural and functional genetic elements mainly derived from retroviruses. The term "lentiviral vector" refers to a vector that includes structural and functional genetic elements mainly from lentiviruses outside of LTR.

[0091] The retroviral vector used in this article can be derived from any known retrovirus (e.g., type c retrovirus, 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), foamy virus, Friend, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV)). The "retrovirus" of the present invention also includes human T-cell leukemia virus, HTLV-1 and HTLV-2, and the lentivirus family of retroviruses, such as human immunodeficiency virus (HIV-1, HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV) and other types of retroviruses.

[0092] As used herein, a lentiviral vector refers to a vector derived from a lentivirus, a class (or genus) of retroviruses that cause a slowly progressing disease. The viruses included in this class include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); progressive interstitial pneumonia of sheep (visna-maedi); caprine arthritis-encephalitis virus; equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). The preparation of recombinant lentiviruses can be achieved using methods according to 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).

[0093] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present invention can be made 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. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; 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 WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573).

[0094] Suitable sources for obtaining retroviral (ie, both lentiviral and non-lentiviral) sequences for forming vectors include, for example, genomic RNA and cDNA, which are available from commercial sources including the American Type Culture Collection (ATCC), Rockville, Md. The sequences may also be chemically synthesized.

[0095] In order to express BCMA CAR, the vector can be introduced into the host cell to allow the polypeptide to be expressed in the host cell. The expression vector may include a variety of elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection markers and signal sequences. As described above, these elements can be appropriately selected by those of ordinary skill in the art. For example, a promoter sequence can be selected to promote transcription of polynucleotides in the vector. Suitable promoter sequences include but are not limited to T7 promoters, T3 promoters, SP6 promoters, beta-actin promoters, EF1a promoters, CMV promoters and SV40 promoters. Enhancer sequences can be selected to enhance transcription of polynucleotides. Selection markers can be selected to allow host cells without insertion of the vector to be selected from those vectors, for example, the selection marker can be a gene that confers antibiotic resistance. A signal sequence can be selected so that the expressed polypeptide is transported outside the host cell.

[0096] In order to clone a polynucleotide, a vector can be introduced into a host cell (isolated host cell) to allow the vector to replicate itself, thereby increasing the copies of the polynucleotide contained therein. The cloning vector can contain sequence components, generally including but not limited to a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selective marker. These elements can be appropriately selected by a person of ordinary skill in the art. For example, a replication origin can be selected to promote autonomous replication of the vector in a host cell.

[0097] In certain embodiments, the disclosure provides a host cell comprising the separation of a vector provided herein. The host cell comprising the vector can be used for expressing or cloning the polynucleotides included in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells or higher eukaryotic cells, such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, true bacteria, such as gram-negative or gram-positive organisms, such as enterobacteria (Enterobactehaceae), such as Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans and Shigella, and bacillus, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas, such as Pseudomonas aeruginosa and Streptomyces.

[0098] The CAR of the present invention is introduced into host cells using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process of introducing exogenous nucleic acid sequences into host cells. The nucleic acid may be integrated into the host cell DNA or may be maintained outside the chromosome. The nucleic acid may be transiently maintained or may be stably introduced. Transfection may be accomplished in a variety of ways known in the art, including but not limited to calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polyene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene gun. Transduction refers to the use of a virus or retroviral vector to deliver a gene by viral infection rather than by transfection. In certain embodiments, a retroviral vector is transduced by packaging it into a virion before contacting the cell. For example, a nucleic acid encoding BCMA CAR carried by a retroviral vector may be transduced into a cell by infection and proviral integration.

[0099] As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell. The terms "genetically modified cell," "modified cell," and "redirected cell" are used interchangeably.

[0100] In particular, the CAR of the present invention is introduced and expressed in immune effector cells in order to redirect their specificity to the target antigen of interest, such as malignant plasma cells, such as multiple myeloma.

[0101] The present invention provides a method for preparing immune effector cells expressing CAR as described herein. In one embodiment, the method includes transfection or transduction of immune effector cells separated from a subject (e.g., a subject of a tumor cell with BCMA expression), so that the immune effector cells express one or more CARs as described herein. In certain embodiments, immune effector cells are separated from an individual and can be genetically modified without further in vitro operation. Such cells can then be directly re-administered to an individual. In other embodiments, before being genetically modified to express CAR, immune effector cells are first activated and stimulated to proliferate in vitro. In this regard, immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express CAR as described herein).

[0102] Cell sources can be obtained from subjects before in vitro manipulation or genetic modification of immune effector cells as described herein. In particular, immune effector cells used with CAR as described herein include T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue and tumor. In certain embodiments, many techniques known to those skilled in the art (such as FICOLL separation) can be used to obtain T cells from unit blood collected from subjects. In one embodiment, cells from individual circulating blood are obtained by apheresis. Apheresis products generally include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In one embodiment, cells collected by apheresis can be washed to remove plasma fractions and cells are placed in appropriate buffers or culture media for subsequent treatment. In one embodiment of the present invention, cells are washed with PBS. In another embodiment, the washed solution lacks calcium, and may lack magnesium, or may lack many (if not all) divalent cations. As will be appreciated by one of ordinary skill in the art, the washing steps can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic flow-through centrifuge. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, unwanted components of the apheresis sample can be removed in the medium in which the cells are directly resuspended.

[0103] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes (e.g., by PERCOLL TM Gradient centrifugation). Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further separated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be accomplished by a combination of antibodies against surface markers specific to negatively selected cells. One method used herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, in order to enrich CD4+ cells by negative selection, a mixture of monoclonal antibodies typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to separate cell populations of interest for use in the present invention.

[0104] Using the methods described herein, PBMC can be directly used for the genetic modification of CAR. In some embodiments, after separating PBMC, T lymphocytes are further separated, and in some embodiments, cytotoxic and helper T lymphocytes can be classified into primary, memory and effector T cell subsets before or after genetic modification and / or amplification. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further classified into primary, central memory and effector cells by identifying cell surface antigens associated with each of those types of CD8+ cells. In embodiments, memory T cells are present in CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are divided into CD62L-CD8+ and CD62L+CD8+ parts. In some embodiments, the expression of phenotypic markers of central memory TCM 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 positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.

[0105] In certain embodiments, CD4+T cells are further classified into subgroups. For example, CD4+T helper 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.

[0106] Immune effector cells, such as T cells, can be genetically modified after being separated using known methods, or before being genetically modified, immune effector cells can be activated and amplified in vitro (or differentiated in the case of progenitor cells). In another embodiment, immune effector cells, such as T cells, are genetically modified with chimeric antigen receptors as described herein (e.g., transduced with a viral vector including a nucleic acid encoding CAR), and then activated and amplified in vitro. Methods for activating and amplifying T cells are known in the art and are described in, for example, U.S. Patent No. 6,905,874; U.S. Patent No. 6,867,041; U.S. Patent No. 6,797,514; WO2012079000. Typically, such methods include: in a culture medium with appropriate cytokines (e.g., IL-2), PBMC or separated T cells are contacted with stimulants and co-stimulators (e.g., anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces). Anti-CD3 and anti-CD28 antibodies attached to the same beads act as "alternative" antigen presenting cells (APCs). In other embodiments, T cell proliferation may be activated and stimulated using feeder cells and appropriate antibodies and cytokines using methods such as those described in US Pat. No. 6,040,177; US Pat. No. 5,827,642; and WO2012129514.

[0107] The present invention provides a population of modified immune effector cells for use in treating patients suffering from malignancies arising from BCMA-expressing tumors (e.g., multiple myeloma), wherein the modified immune effector cells include a BCMA CAR disclosed herein.

[0108] The immune effector cells expressed by CAR prepared as described herein can be used in methods and compositions of adoptive immunotherapy according to known techniques, or variations thereof that are apparent to those skilled in the art based on the present disclosure. See, e.g., U.S. Patent Application Publication No. 2003 / 0170238 of Gruenberg et al.; see also U.S. Patent No. 4,690,915 of Rosenberg.

[0109] In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a therapeutically effective amount of a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate can also be used. The infusion medium can be supplemented with human serum albumin.

[0110] The therapeutically effective amount of cells in the composition is at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset), or more usually greater than 10 2 cells, up to 10 6 Up to and including 10 8 or 10 9 cells, and can be greater than 10 10 The number of cells will depend on the intended end use of the composition, as well as the type of cells contained therein.

[0111] Cells can be autologous or allogeneic to the patient being treated. If necessary, as described herein, treatment also includes 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.) to enhance the induction of immune responses.

[0112] The immune effector cell colonies expressing CAR of the present invention can be administered alone, or as a pharmaceutical composition and diluent and / or in combination with other components such as IL-2 or other cytokines or cell colonies. In short, the pharmaceutical composition of the present invention may include immune effector cell colonies expressing CAR, such as T cells as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0113] The anti-tumor immune response induced in a subject by administering the T cells expressed by the CAR described herein using the methods described herein or other methods known in the art may include cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, regulatory T cells, and helper T cell responses. It is also possible to induce a humoral immune response mediated mainly by helper T cells capable of activating B cells to cause antibody production. A variety of techniques can be used to analyze the type of immune response induced by the composition of the present invention, which has been well described in the art; for example, Current Protocols in Immunology, editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0114] Thus, the present invention provides a method for treating an individual diagnosed with or 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 (e.g., in multiple myeloma), comprising administering to the individual a therapeutically effective amount of a CAR-expressing immune effector cell described herein.

[0115] In one embodiment, the present invention provides a method for treating a subject diagnosed with a BCMA-expressing cancer, the method comprising removing immune effector cells from a subject diagnosed with a BCMA-expressing cancer, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding a chimeric antigen receptor of the present invention, thereby producing a modified immune effector cell population, and administering the modified immune effector cell population to the same subject. In one embodiment, the immune effector cells include T cells.

[0116] The method for administering a cell composition as described herein includes any method effectively causing reintroduction of an in vitro genetically modified immune effector cell, the immune effector cell directly expresses the CAR of the present invention in the subject or when reintroducing the genetically modified progenitor cell of the immune effector cell (differentiating into mature immune effector cells expressing CAR after introducing the subject) expression. A method includes ex vivo transduction of peripheral blood T cells with a nucleic acid construct according to the present invention, and returning the transduced cells to the subject.

[0117] Binding properties of chimeric antigen receptors and corresponding antibodies

[0118] As used herein, the term "binding" in the context of binding of a chimeric antigen receptor or a corresponding antibody to, for example, a predetermined antigen (such as a cell surface protein) or a fragment thereof, generally refers to an interaction or association between at least two entities or molecular structures, such as an antigen binding domain:antigen interaction.

[0119] For example, when measured in a BIAcore 3000 instrument using antigen as ligand and antibody or chimeric antigen receptor as analyte (or anti-ligand) by, for example, surface plasmon resonance (SPR) technology, binding affinity typically corresponds to about 10 -7 M or smaller KD value, such as 10 -8 M or smaller, such as about 10 -9 M or less. Cell-based binding strategies, such as fluorescence activated cell sorting (FACS) binding assays, are also commonly used, and FACS data correlate strongly with other methods, such as radioligand competition 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).

[0120] Thus, the chimeric antigen receptor or corresponding antibody of the present invention binds to a predetermined antigen or cell surface molecule (receptor) having a corresponding K D According to the present invention, the K of a chimeric antigen receptor or a corresponding antibody is at least ten times lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). D Affinity values ​​equal to or less than ten times that of the nonspecific antigen can be considered as undetectable binding.

[0121] The term "K D "(M)" refers to the dissociation equilibrium constant of a specific antigen binding domain:antigen interaction, or the dissociation equilibrium constant of the corresponding antibody and antigen. K D There is an inverse relationship between K and binding affinity, so K D The smaller the value, the higher the affinity, ie the stronger it is. Therefore, the term "higher affinity" or "stronger affinity" relates to a higher ability to form an interaction and therefore to a smaller K D value, and conversely, the term "lower affinity" or "weaker affinity" relates to a lower ability to form an interaction, and thus K D In some cases, the binding affinity (or K) of a particular molecule (e.g., a chimeric antigen receptor or a corresponding antibody) to its interacting partner molecule (e.g., antigen X) is D) is higher than the binding affinity of the molecule (e.g., chimeric antigen receptor or corresponding antibody) to another interaction partner molecule (e.g., antigen Y), and the higher binding affinity can be expressed by dividing the larger K D value (lower or weaker affinity) divided by the smaller K D The binding rate may be determined based on a higher or stronger affinity, for example, expressed as a 5-fold or 10-fold greater binding affinity, as the case may be.

[0122] The term “k d "(sec-1 or 1 / s) refers to the dissociation rate constant for a specific antigen binding domain:antigen interaction, or the dissociation rate constant for a chimeric antigen receptor or corresponding antibody. This value is also referred to as k off value.

[0123] The term “k a "(M-1 x sec-1 or 1 / M) refers to the association rate constant for a specific antigen binding domain:antigen interaction, or the association rate constant for a chimeric antigen receptor or corresponding antibody.

[0124] The term "K A "(M-1 or 1M) refers to the association equilibrium constant of a specific antigen binding domain:antigen interaction, or the association equilibrium constant of a chimeric antigen receptor or corresponding antibody. k a Divide by k d The association equilibrium constant was obtained.

[0125] The term "EC50" or "EC 50 ” refers to the half maximal effective concentration, which includes the concentration of chimeric antigen receptor that induces a response halfway between baseline and maximum after a specified exposure time. EC 50 represents substantially the concentration of the chimeric antigen receptor at which 50% of its maximal effect is observed. 50 The value is equal to the concentration of the chimeric antigen receptor or corresponding antibody of the invention that confers half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen such as BCMA), as determined by, for example, a FACS binding assay. Thus, decreased or weaker binding is observed, said binding having an increased EC 50 Or half of the maximum effective concentration.

[0126] In one embodiment, decreased binding can be defined as increased EC 50 The concentration of chimeric antigen receptor or corresponding antibody that is able to bind to half the maximal number of target cells.

[0127] Sequence variants of chimeric antigen receptors

[0128] The chimeric antigen receptor or the present invention may include 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 of the respective antigen binding domains from which the corresponding antibodies are derived. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained, for example, from public antibody sequence databases. The chimeric antigen receptor of the present invention may include an antigen binding domain derived from any exemplary CDR or variable region amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding one or more residues of the germline sequence from which the corresponding antibody is derived, or mutated to the corresponding one or more residues of another human germline sequence, or mutated to the corresponding conservative amino acid substitution of one or more germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, a person of ordinary skill in the art can easily produce many antibodies and antigen binding fragments including one or more individual germline mutations or combinations thereof. In some instances, V H Domain and / or V L In some embodiments, the framework and / or CDR residues in the domain are all mutated back to the residues found in the original germline sequence of the initial derived antigen-binding domain. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only the residues after the mutation found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only the residues after the mutation found in CDR1, CDR2 or CDR3. In other examples, one or more mutations in the framework and / or one or more CDR residues are one or more corresponding residues of different germline sequences (that is, germline sequences different from the germline sequences of the initial derived antigen-binding domains). In addition, the antigen-binding domains of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, wherein some individual residues are mutated into the corresponding residues of a specific germline sequence, and some other residues different from the original germline sequence can be maintained or mutated into the corresponding residues of different germline sequences.

[0129] Biological properties of chimeric antigen receptors and corresponding antibodies

[0130] The present invention includes chimeric antigen receptors having an antigen binding domain derived from a polypeptide that binds with high affinity (e.g., nanomolar or subnanomolar K D Value) Antibodies that bind to human BCMA.

[0131] According to certain embodiments, the invention comprises a chimeric antigen receptor having an antigen binding domain derived from a polypeptide having a K of less than about 5 nM as measured by surface plasmon resonance. DIn some embodiments, the corresponding antibody has a K D Binding to BCMA: 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.

[0132] The present invention also includes a chimeric antigen receptor having an antigen binding domain derived from a chimeric antigen receptor having a dissociation half-life (t 1 / 2) a corresponding antibody that binds BCMA. In certain embodiments, the corresponding antibody is expressed in such a manner that 1 / 2 binding to BCMA: 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.

[0133] The 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.

[0134] The present invention also includes engineered cells expressing a BCMA-specific chimeric antigen receptor that (i) is activated by BCMA-expressing cells, and / or (ii) exhibits an inhibitory effect on tumor growth in immunocompromised mice bearing human multiple myeloma xenografts.

[0135] Preparation of antigen binding domain

[0136] The antigen binding domain of the chimeric antigen receptor of the present invention that is 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 individual components (e.g., heavy and light chains) of the corresponding antibodies of the present invention are derived from chimeric, humanized, or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMMUNE can be used TMOne or more heavy chains and / or light chains are prepared using VELOCIMMUNE TM The technique (or any other human antibody production technique) first isolates high-affinity chimeric antibodies against a specific antigen (e.g., BCMA) with human variable regions and mouse constant regions. The antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. As described herein, these human variable regions (or CDRs) can then be incorporated into the antigen binding domain of a chimeric antigen receptor.

[0137] Polynucleotides and vectors

[0138] The present invention also relates to polynucleotides and vectors encoding the chimeric antigen receptors discussed herein.

[0139] In various embodiments, the polynucleotide can include an expression cassette or an 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).

[0140] In various embodiments, the polynucleotide and / or vector comprises a nucleic acid molecule comprising a 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 an 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 an amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100.

[0141] Methods for engineering immune cells expressing chimeric antigen receptors

[0142] The present invention includes methods of preparing immune cells for use in immunotherapy, the methods comprising introducing into such immune cells ex vivo a polynucleotide or vector encoding one of the BCMA-specific chimeric antigen receptors described herein.

[0143] The present invention also encompasses immune cells comprising a polynucleotide or lentiviral vector encoding one of the BCMA-specific chimeric antigen receptors discussed herein. In some embodiments, these immune cells are used for immunotherapy (eg, treatment of cancer).

[0144] The present invention also includes a method for genetically modifying immune cells to make them more suitable for allogeneic transplantation. According to a first aspect, immune cells can be made allogeneic, for example, by inactivating the genes of at least one or more components expressing a T cell receptor (TCR), as described in WO 2013 / 176915, which can be combined with the inactivation of genes encoding or regulating HLA or β2m protein expression. Therefore, the risk of graft-versus-host syndrome and graft rejection is significantly reduced. According to another aspect of the present invention, immune cells can be further manipulated to make them more active or limit exhaustion by inactivating the genes encoding proteins acting as "immune checkpoints" (acting as regulators of T cell activation, such as PD1 or CTLA-4).

[0145] Engineered immune cells

[0146] The immune cell (or engineered immune cell) comprising the chimeric antigen receptor of the present invention is another object of the present invention. In some cases, the immune cell is an immune effector cell. In some cases, the immune cell is a T cell. In some cases, the immune cell is a T lymphocyte selected from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes. In some cases, the immune cell is a CD8+ cytotoxic T lymphocyte.

[0147] In some embodiments, the engineered immune cell is a human T cell comprising a chimeric antigen receptor, which comprises, from N-terminus to C-terminus: (a) an extracellular ligand binding domain comprising an anti-BCMA single-chain variable fragment (scFv) domain, which includes 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 co-stimulatory domain and a signaling domain.

[0148] In some embodiments, the scFv domain of the engineered human T cell comprises a LCVR / HCVR amino acid sequence pair comprising an amino acid sequence of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50, or 74 / 66. In some cases, the hinge comprises an amino acid sequence of SEQ ID NO: 97. In some cases, the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 98. In some cases, the costimulatory domain is a 4-1BB costimulatory domain. In some cases, the 4-1BB costimulatory domain comprises an amino acid sequence of SEQ ID NO: 99. In some cases, the signaling domain is a CD3zeta signaling domain. In some cases, the CD3zeta signaling domain comprises an amino acid sequence of SEQ ID NO: 100.

[0149] In various embodiments, the engineered human T cell comprises 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.

[0150] Bioequivalent

[0151] The present invention includes chimeric antigen receptors and engineered cells expressing chimeric antigen receptors, which have amino acid sequences different from those of the exemplary molecules disclosed herein, but retain the following abilities: bind BCMA; activate immune cells expressing chimeric antigen receptors in the presence of BCMA expressing cells; or inhibit the growth or proliferation of BCMA expressing tumor cells. Such variant molecules may include one or more additions, deletions or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity substantially the same as that of the bispecific antigen binding molecule.

[0152] In one embodiment, two engineered immune cells expressing a chimeric antigen receptor of the invention are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0153] In one embodiment, two bioengineered immune cells are bioequivalent if a patient can switch one or more times between the reference product and the biological product without an expected increase in risk of adverse effects, including a clinically significant change in immunogenicity or a decrease in effectiveness compared to continued treatment without such switching.

[0154] In one embodiment, two engineered immune cells are bioequivalent if they both act by a common mechanism for one or more conditions of use (to the extent such mechanism is known).

[0155] Bioequivalence can 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 in blood, plasma, serum or other biological fluids is varied over time; (b) in vitro tests that are correlated with and reasonably predictive of human in vivo bioavailability data; (c) in vivo tests in humans or other mammals, where appropriate acute pharmacological effects of the engineered cells (or their targets) are measured over time; and (d) in well-controlled clinical trials that establish the safety, efficacy, bioavailability or bioequivalence of the engineered cells.

[0156] Bioequivalent variants of the exemplary engineered cells described herein can be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not required for biological activity.

[0157] Species selectivity and cross-species reactivity

[0158] According to certain embodiments of the present invention, an antigen binding domain is provided that binds to human BCMA but not to BCMA of other species. The present invention also includes an antigen binding domain that binds to human BCMA and BCMA of one or more non-human species.

[0159] According to certain exemplary embodiments of the invention, there are provided antigen binding domains that bind to human BCMA, and may or may not bind to mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, macaque, marmoset, rhesus monkey, or chimpanzee BCMA, as the case may be.

[0160] Activation and expansion of engineered immune cells

[0161] Whether before or after genetic modification of engineered cells (e.g., T cells), even if the genetically modified immune cells of the present invention are activated and proliferate independently of the antigen binding mechanism, the immune cells of the present invention (particularly T cells) can generally be further activated and expanded using methods described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

[0162] Typically, the T cells of the present invention are expanded by contacting with agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of the T cells to generate activation signals for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA) or phytohemagglutinin (PHA) such as mitogen can be used to generate activation signals for T cells.

[0163] As a non-limiting example, T cell colonies can be stimulated in vitro, for example, by contacting with anti-CD3 antibodies or their antigen binding fragments or anti-CD2 antibodies fixed on the surface, or by contacting a protein kinase C activator (e.g., a cytostatin) bound to a calcium ion carrier. In order to costimulate auxiliary molecules on the T cell surface, a ligand binding auxiliary molecule is used. For example, under conditions suitable for stimulating T cell proliferation, a T cell colony is contacted with an anti-CD3 antibody and an anti-CD28 antibody. Conditions suitable for T cell culture include appropriate culture media (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 5, (Lonza)), which may include factors required for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin 2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp and TNF-α or any other additives known to technicians for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma compounds, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoglycolic acid. The medium may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer, with the addition of amino acids, sodium pyruvate, and vitamins (without serum) or supplemented with an appropriate amount of serum (or plasma) or a set of determined hormones, and / or cytokines sufficient to grow and amplify T cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures to be injected into the subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% O2). T cells exposed to different stimulation times may exhibit different characteristics.

[0164] In another specific embodiment, the cells can be expanded by co-culturing with tissues or cells. The cells can also be administered in vivo, for example, after the cells are administered to a subject, they expand in the blood of the subject.

[0165] Therapeutic applications

[0166] The present invention includes compositions comprising engineered cells (e.g., T cells) expressing chimeric antigen receptors of the present invention and a pharmaceutically acceptable vehicle. In some cases, engineered cells form drugs, particularly for immunotherapy. In some cases, engineered cells are used to treat cancer (e.g., multiple myeloma). In some cases, engineered cells are used to manufacture drugs for immunotherapy and / or treatment of cancer (e.g., cancers expressed by BCMA).

[0167] The present invention includes such a method, which includes administering a therapeutic composition to a subject in need thereof, the therapeutic composition including engineered cells (e.g., T cells) expressing a chimeric antigen receptor described herein. The therapeutic composition may include cells expressing any chimeric antigen receptor disclosed herein and a pharmaceutically acceptable carrier, diluent, or vehicle. As used herein, the expression "subject in need" refers to a human or non-human animal (e.g., a subject expressing a tumor or suffering from any cancer mentioned herein) that exhibits symptoms or signs of one or more cancers, or otherwise benefits from the inhibition or reduction of BCMA activity or the depletion of BCMA+ cells (e.g., multiple myeloma cells).

[0168] The engineered cells of the present invention are particularly useful for treating any disease or condition where stimulation, activation and / or targeting of an immune response would be beneficial. In particular, the engineered cells of the present invention can be used to treat, prevent and / or ameliorate any disease or condition associated with or mediated by BCMA expression or activity or BCMA+ cell proliferation. Cells expressing BCMA that can be inhibited or killed using the engineered cells of the present invention include, for example, multiple myeloma cells.

[0169] The engineered cells of the invention can be used to treat diseases or conditions associated with BCMA expression, such as cancers including multiple myeloma or other B cell or plasma cell cancers, e.g. Macroglobulinemia, Burkitt's lymphoma and diffuse large B-cell lymphoma. In some embodiments, the disease or condition in which BCMA is expressed is Castleman's disease, lymphoplasmacytic 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 can be used to treat patients with multiple myeloma. According to other related embodiments of the present invention, there is provided a method comprising administering an engineered cell as disclosed herein to a patient with multiple myeloma. Analysis / diagnostic methods known in the art, such as tumor scanning, etc., can be used to determine whether a patient has multiple myeloma or another B-cell lineage cancer.

[0170] The present invention also includes methods for treating residual cancer in a subject.As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0171] According to certain aspects, the present invention provides methods for treating a disease or condition associated with BCMA expression (e.g., multiple myeloma), comprising administering to a subject an engineered cell population described elsewhere herein after determining that the subject suffers from multiple myeloma. For example, the present invention includes methods for treating multiple myeloma, comprising administering engineered immune cells to a patient for 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 receives other immunotherapy or chemotherapy.

[0172] The treatments discussed herein may be ameliorative, curative, or preventative. Treatment may be part of an autologous immunotherapy or part of an allogeneic immunotherapy. Autologous means that the cells, cell line, or cell population used to treat the patient are derived from the patient or a human leukocyte antigen (HLA) compatible donor. Allogeneic means that the cells, cell line, or cell population used to treat the patient do not originate from the patient but from a donor.

[0173] Cells that can be used with the disclosed methods are described herein. The therapy can be used to treat patients diagnosed with a pre-malignant or malignant cancer condition characterized by BCMA-expressing cells, particularly patients characterized by an excess of BCMA-expressing cells. This condition is found in cancers such as multiple myeloma.

[0174] The types of cancers treated with the engineered cells of the present invention include, but are not limited to, multiple myeloma, In some embodiments, the engineered cells can be used to treat diseases or conditions in which BCMA is expressed, 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.

[0175] The compositions and methods of the invention can be used to treat subjects that have been characterized as having cells or tissues expressing BCMA, or suspected of having cells or tissues expressing BCMA. For example, subjects that benefit from treatment according to the invention include subjects with multiple myeloma.

[0176] Administration of cells or cell populations according to the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. Compositions as described herein can be administered to patients by intravenous or intralymphatic injection or intraperitoneal, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, transdermal administration. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0177] Administration of cells or cell populations may include administration of 10 4 -10 9 cells, preferably 10 5 Up to 10 6 Cells / kg body weight, including integer values ​​of all cell numbers within those ranges. Cells or cell colonies can be administered in one or more doses. In some embodiments, an effective amount of cells is administered in a single dose. In some embodiments, an effective amount of cells is administered in a dose of more than one dose over a period of time. The administration time is within the physician's discretion and depends on the patient's clinical condition. Cells or cell colonies can be obtained from any source, such as a blood bank or a donor. Although individual needs vary, the determination of the effective amount range for a given cell type for a specific disease or condition is within the technical scope of the art. An effective amount refers to an amount that provides a therapeutic or preventive benefit. The dosage administered will depend on the recipient's age, health status and weight, the type of treatment being performed simultaneously (if any), the frequency of treatment and the nature of the desired effect.

[0178] In one embodiment, an effective amount of cells or a composition comprising those cells is administered parenterally. The administration can be intravenous. In some cases, administration can be performed directly by injection into the tumor.

[0179] In certain embodiments of the invention, the cells are administered to a patient in conjunction with (e.g., before, at the same time, or after) any number of related treatment modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, treatment of MS patients with cytarabine (also known as ARA-C) or natalizumab, treatment of psoriasis patients with efaliximab, or other treatments for PML patients. In other embodiments, the T cells of the invention can be used in conjunction with chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents (e.g., CAMPATH, anti-CD3 antibodies) or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycoplasma acid, steroids, FR901228, cytokines, and radiation.

[0180] In another embodiment, the cell composition of the present invention is administered to the patient in combination with (e.g., before, at the same time, or after) bone marrow transplantation, T cell ablation therapy using any chemotherapeutic agent (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (e.g., OKT3 or CAMPATH). In another embodiment, the cell composition of the present invention is administered after B cell ablation therapy (e.g., an agent that reacts with CD20, such as rituximab). For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the amplified immune cells of the present invention. In another embodiment, the amplified cells are administered before or after surgery. In certain embodiments, before administering the amplified immune cells of the present invention, any means (e.g., surgery, chemotherapy, or radiotherapy) may be used to reduce tumor burden. In one embodiment, reducing tumor burden before administering the engineered cells of the present invention can reduce or prevent the possibility of cytokine release syndrome or cytokine storm, or prevent side effects associated with CAR T cell therapy.

[0181] Combination therapy

[0182] The present invention provides methods including administering engineered cells or cell groups including any chimeric antigen receptor described herein in combination with one or more other therapeutic agents. Other exemplary therapeutic agents that can be combined or co-administered with the cells or cell colonies of the present invention include, for example, anti-tumor agents (e.g., chemotherapeutic agents, including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), or any other therapeutic agent known to effectively treat plasma cell tumors in subjects). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes a targeted therapeutic agent, including thalidomide, lenalidomide, and bortezomib, which are therapeutic agents approved for the treatment of newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, alozumab, and daratumumab are examples of second therapeutic agents that are effective in treating relapsed myeloma. In some embodiments, the second therapeutic agent is a scheme including radiotherapy or stem cell transplantation. In some embodiments, the second therapeutic agent can be an immunomodulator. In some embodiments, the second therapeutic agent can be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In some embodiments, the second therapeutic agent can be a histone deacetylase inhibitor, such as panobinostat (Farydak). In some embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof. Other agents that can be beneficially administered in combination with the antigen binding molecules of the present invention include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies bound 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 their respective receptors. The pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising engineered cells or cell populations disclosed herein) can also be administered as part of a treatment regimen comprising one or more therapeutic combinations of monoclonal antibodies other than those described herein that can interact with: another antigen on the surface of plasma cells; a bispecific antibody, one arm of which binds to an antigen on the surface of a tumor cell and the other arm binds to an antigen on a T cell; an antibody-drug conjugate; a bispecific antibody coupled to an anti-tumor agent; a checkpoint inhibitor (e.g., an antibody targeting PD-1 or CTLA-4); or a combination thereof. In certain embodiments, the checkpoint inhibitor can be selected from a PD-1 inhibitor, such as pembrolizumab (Keytruda), nivolumab (Opdivo), or semipirimab (REGN2810).In certain embodiments, the checkpoint inhibitor can be selected from a PD-L1 inhibitor, such as atezolizumab (Tecentriq), avacumab (Bavencio), or duruvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor can be selected from a CTLA-4 inhibitor, such as ipilimumab (Yervoy).

[0183] The present invention also includes therapeutic compositions, which include any engineered cells or cell populations mentioned herein and 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 above cytokines One or more inhibitors, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, siRNA, a peptibody, a nanobody or an antibody fragment (e.g., a Fab fragment; a F(ab')2 fragment; a Fd fragment; a Fv fragment; a scFv; a dAb fragment; or other engineered molecules such as double antibodies, three antibodies, four antibodies, micro antibodies and minimal recognition units). In some embodiments, the engineered cells or cell populations of the present invention can also be administered as part of a treatment regimen that also includes radiotherapy and / or conventional chemotherapy.

[0184] One or more additional therapeutically active components may be administered prior to, concurrently with, or after administration of the engineered cells of the invention; (for purposes of this disclosure, such administration regimens are considered administration of the engineered cells "in combination" with the additional therapeutically active components).

[0185] The invention includes pharmaceutical compositions in which the engineered cells or cell populations of the invention are co-formulated with one or more additional therapeutically active ingredients as described elsewhere herein.

[0186] Dosage regimen

[0187] According to certain embodiments of the present 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 present invention includes administering multiple doses of cells to a subject sequentially. As used herein, "sequential administration" means that each dose is administered to a subject at different time points, for example, on different dates separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes the following method: it includes administering a single initial dose to a patient sequentially, followed by one or more second doses, and optionally subsequently administering one or more third doses.

[0188] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time sequence of administration of the engineered cells of the present invention. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary dose" is the dose administered after the initial dose; and the "tertiary dose" is the dose administered after the second dose. The initial dose, the second dose, and the third dose may all contain the same amount of engineered cells, but may generally differ from each other in terms of the frequency of administration. However, in certain embodiments, during the course of treatment, the amount of engineered cells contained in the initial dose, the second dose, and / or the third dose differs from each other (e.g., appropriately adjusted up or down). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0189] In an exemplary embodiment of the invention, each second dose and / or third dose is administered 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, 10 1 / 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, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 As used herein, the phrase "previous dose" means a dose administered to a patient in a sequence of multiple doses that is administered to the patient prior to the next dose in the sequence, with no intervening doses.

[0190] The method according to this aspect of the invention may include administering any number of second and / or third doses to the patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient.

[0191] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as other third doses. For example, each third dose can be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency of administering the second dose and / or the third dose to the patient can change during the course of the treatment regimen. The frequency of administration can also be adjusted during the physician's treatment according to the needs of individual patients after clinical examination.

[0192] Examples

[0193] The following examples are presented to provide a complete disclosure and description of how to prepare and use the methods and compositions of the present invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is or is close to atmospheric pressure.

[0194] Example 1: Generation of anti-BCMA antibodies

[0195] Anti-BCMA antibodies are obtained by immunizing genetically modified mice with human BCMA antigen (e.g., hBCMA, SEQ ID NO: 101), or by immunizing engineered mice comprising DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions with human BCMA antigen.

[0196] After immunization, spleen cells were harvested from each mouse and either (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells and screened for BCMA specificity, or (2) B cells were sorted using human BCMA fragments as sorting agents to bind and identify reactive antibodies (antigen-positive B cells) (as described in US2007 / 0280945A1).

[0197] Initially isolated are chimeric antibodies against BCMA with human variable regions and mouse constant regions. The antibodies are characterized and selected to obtain desired properties including affinity, selectivity, etc. If necessary, the mouse constant region is replaced with a desired human constant region (e.g., a wild-type or modified IgG1 or IgG4 constant region) to produce a fully human anti-BCMA antibody. Although the selected constant region can vary depending on the specific use, high affinity antigen binding and target specificity characteristics are present in the variable region.

[0198] The amino acid and nucleic acid sequences of the heavy and light chain variable regions of the anti-BCMA antibodies: Table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the anti-BCMA antibodies selected in the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0199] Table 1: Amino Acid Sequence Identifiers

[0200]

[0201] Table 2: Nucleotide sequence identifiers

[0202]

[0203]

[0204] Example 2: Generation of BCMA-specific chimeric antigen receptors

[0205] Six anti-BCMA antibodies (mAb16711, mAb16716, mAb16732, mAb16747, and mAb21581) were reformatted into VL-VH single-chain variable fragments (ScFv) and placed into chimeric antigen receptor (CAR) constructs using the CD8α hinge and transmembrane domains, the 4-1BB co-stimulatory domain, and the CD3ζ stimulatory domain. The BCMA-specific CAR was cloned into a lentiviral expression vector (Lenti-X TM Bicistronic Expression System (Neo, Clontech Cat#632181) and generated lentiviral particles using Lenti-X Packaging Single-Shot (VSV-G) System (Clontech Cat#631276) according to the manufacturer's protocol. Precoated Dishes (Clontech, Cat#T110a) were transduced with different CAR constructs into Jurkat cells (Jurkat / NFATLuccl.3C7) engineered to express a NFAT luciferase reporter. After selection in 500 μg / ml G418 (Gibco, Cat#11811-098) for at least 2 weeks, the following CAR-T cell lines were generated; Jurkat / NFATLuc cl.3C7 / BCMA 16716VL-VH CART, Jurkat / NFATLuc cl.3C7 / BCMA 16711VL-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. Figure 1 As shown, 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), 83 (mAb16716 VL / VH), 85 (mAb16732 VL / VH), 87 (mAb16747 VL / VH) and 89 (mAb21581 VL / VH). As described in Example 3, these six CAR-T cell lines were used to evaluate the cell surface expression and activation of BCMA CAR-T cells.

[0206] The VL and VH nucleotide sequences of two anti-BCMA antibodies mAb21581 and mAb16747 (corresponding to SEQ ID NO: 89 and 87, respectively) were used to construct a chimeric antigen receptor comprising an anti-BCMA VL-VH scFv, a huCD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. As a non-binding control, a similar CAR was designed using the nucleotide sequence of an unrelated scFv (SEQ ID NO: 91 CAR construct). These CARs were cloned into a pLVX lentiviral vector with an EF1a promoter and an IRES: eGFP sequence (for tracking CAR-transduced cells), and VSV pseudotype lentivirus was produced.

[0207] CD3+T cells were isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads plus 100U / ml recombinant human IL-2, and transduced with lentivirus at MOI=5. The transduced cells were expanded for 3 weeks with CD3 / CD28 microbeads plus 100U / ml recombinant human IL-2 and then cryopreserved until used in in vivo experiments. As described in Examples 4 and 5, these three lines of CAR-T cells were used to evaluate the efficacy of reducing tumor burden in vivo.

[0208] Example 3: Cell surface expression of BCMA CAR constructs in Jurkat cells and activation of BCMA CAR-T cells

[0209] The relative cell surface expression of BCMA CAR constructs in Jurkat / NFATLuc cells was obtained by flow cytometry. For staining, cells were seeded in a 96-well V-bottom plate at a density of 200,000 cells per well in a staining buffer (PBS, calcium and magnesium-free (Irving 9240) + 2% FBS (ATCC 30-2020)), and stained at 4 ° C with 10ug / ml of BCMA extracellular domain (BCMA ecto-hFc) fused to hIgG1-Fc or an unrelated protein (Fc isotype control) fused to hIgG1-Fc for 30min. After incubation with BCMA-hFc or Fc isotype controls, the cells were washed once in staining buffer and stained at 4 ° C with 10 μg / ml of a secondary antibody coupled to Alexa-Flour 647 (Jackson ImmunoResearch, Cat#109-606-170) for 30min. The cells were then washed and fixed using a 50% BD Cytofix (BD, Cat#554655) solution diluted in staining buffer. The samples were run on an Intellicyt iQue flow cytometer and analyzed by FlowJo 10.2 to calculate the mean fluorescence intensity (MFI). The signal-to-noise ratio (S:N) was determined by the ratio of the BCMA-hFc or Fc isotype control MFI to the secondary antibody MFI alone.

[0210] The activity of the CAR-T system was evaluated in the CAR-T / APC (antigen presenting cell) bioassay. For bioassay, 50,000 CAR-T cells were added to a 50ul assay medium (RPMI medium containing 10% FBS and 1% P / S / G) in a Thermo-Nunc 96-well white plate (Thermo Scientific, Cat#136101), and then a 3-fold serial dilution of APC (500,000 cells to 685 cells) was added to the 50ul assay medium. The following APCs were used: RAJI, Daudi, RPMI8226 (endogenously expressing BCMA) and HEK293 (BCMA negative). The cell mixture was incubated at 37°C, 5% C02, and the incubator was humidified for 5 hours. NFAT-luciferase activity was measured using Promega One-Glo (Cat#E6130) and Perkin Elmer Envision plate readers. Relative luciferase units (RLU) were generated and plotted in GraphPad Prism using a four-parameter logistic equation on an 8-point response curve. The zero APC condition for each dose-response curve was also included in the analysis as a continuation of the three-fold serial dilution and is represented as the lowest dose. CAR-T activity was determined by the ratio of the highest to lowest RLU on the curve and is represented as signal:noise (S:N) in Table 4.

[0211] Table 3 shows that 16747 and 21581 CAR-T have similar surface expression, with S:N ranging from 209-273, 16716 CAR-T has expression 44-fold higher than background, while 16732 and 16711 CAR expression is much lower than S:N, 13 and 4, respectively.

[0212] Table 4 shows that all six BCMA CAR-T cell lines were activated by RAJI, Daudi, and RPMI8226 cells. HEK293 did not activate the CAR-T cell lines. Independent of BCMA expression APC, 16747BCMA CAR had the strongest activation in the CAR-T / APC bioassay, while CAR 16711 had the weakest activity. Finally, a correlation between CAR expression (Table 3) and CAR activity (Table 4) was observed.

[0213] Table 3: Soluble FC-BCMA bound on BCMA CAR-T cell lines

[0214]

[0215]

[0216] Table 4: Activation of BCMA CAR-T in CAR-T / APC Bioassay

[0217]

[0218] Example 4: CAR-T cells targeting BCMA reduce the growth of BCMA-expressing tumors (OPM-2) in vivo in a xenogeneic tumor model

[0219] To determine the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting BCMA, xenograft studies were performed in mice using OPM-2 human multiple myeloma cells that express high levels of BCMA.

[0220] Xenograft tumor implantation and measurements: On day 0, immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were intravenously administered 2x10 6 BCMA + OPM-2 human multiple myeloma tumor cells (OPM-2-luciferase cells). On day 21, mice were injected intravenously with 2x10 6 T cells expressing either a control CAR or an anti-BCMA CAR (determined by the frequency of cells expressing GFP, a marker for those cells that have been transduced with the CAR). Mice (n=5 per group) were dosed with 2x10 6 Irrelevant scFc CAR T (control scFv CAR), 2x10 6 10 anti-BCMA CAR T cells encoding 21581scFv CAR, or 2x10 cells encoding 16747scFv 6 Anti-BCMA CAR T. Tumor growth was assessed by measuring tumor bioluminescence (BLI) in anesthetized animals until day 61. As a positive control, one group of mice (n=5) was administered only OPM-2-luciferase cells without T cells. To measure background BLI levels, one group of mice (n=5) was untreated and did not receive tumors or T cells.

[0221] Measurement of xenograft tumor growth: BLI imaging was used to measure tumor burden. Mice were injected intraperitoneally with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after injection, mice were imaged by BLI using a Xenogen IVIS system under isoflurane anesthesia. Image acquisition was performed with a D field of view, 1.5 cm object height, and medium binning level, with automatic exposure time determined by Living Image Software. BLI signals were extracted using Living Image Software: regions of interest were drawn around each tumor mass and photon intensity was recorded as p / s / cm2 / sr.

[0222] Although BCMA was expressed in mice receiving irrelevant scFv CAR T cells, + OPM-2-luciferase tumors gradually grew, but CAR T cells encoding 21581scFV CAR reduced the tumor burden of most animals to background levels, while CAR T cells encoding 16747scFv CAR reduced the tumor burden of all animals to background levels. The results are shown in Table 5a below.

[0223] Table 5a: Average tumor size at different time points (by radiation)

[0224]

[0225]

[0226]

[0227] Further experiments were performed as described above, except that mice were injected intravenously with 2x10 CAR expressing control CAR or anti-BCMA CAR on day 22 (instead of day 21). 6 T cells, and tumor growth was assessed until day 56 (rather than day 61).

[0228] Although BCMA + OPM-2-luciferase tumors gradually grew in mice receiving irrelevant scFv CAR T cells, but CAR T cells encoding 21581 and 16747 scFV CARs reduced tumor burden to background levels in all animals. The results are shown in Table 5b below.

[0229] Table 5b: Average tumor size at different time points (by radiation)

[0230]

[0231]

[0232]

[0233] Example 5: BCMA-targeted CAR-T cells reduced the growth of BCMA-expressing tumors (MOLP-8) in vivo in a xenogeneic tumor model

[0234] To determine the in vivo efficacy of chimeric antigen receptor (CAR) T cells targeting BCMA, xenograft studies were performed in mice using MOLP-8 human multiple myeloma cells expressing low levels of BCMA.

[0235] Xenograft tumor implantation and measurements: On day 0, the immune-deficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ(NSG) mice were intravenously administered 2x10 6 BCMA + MOLP-8 human multiple myeloma tumor cells (MOLP-8-luciferase cells) engineered to also express firefly luciferase. On day 12, mice were injected intravenously with 2x10 6 T cells expressing either a control CAR or an anti-BCMA CAR (determined by the frequency of cells expressing GFP, a marker for those cells that had been transduced with the CAR). Mice (n=5 per group) received 2x10 6 Irrelevant scFv CAR T (control scFv CAR), 2x10 6 Anti-BCMA CAR T encoding 21581scFv CAR or 2x10 6 Anti-BCMA CAR T encoding 16747scFv. Throughout the experiment, tumor growth was assessed by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a group of mice (n=5) received only MOLP-8-luciferase cell therapy without T cell therapy. To measure background BLI levels, a group of mice (n=5) were untreated and did not receive tumors or T cells.

[0236] Measurement of xenograft tumor growth: BLI imaging was used to measure tumor burden. Mice were injected intraperitoneally with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after injection, mice were imaged by BLI using a Xenogen IVIS system under isoflurane anesthesia. Image acquisition was performed with a D field of view, 1.5 cm object height, and medium binning level, with automatic exposure time determined by Living Image Software. BLI signals were extracted using Living Image Software: regions of interest were drawn around each tumor mass and photon intensity was recorded as p / s / cm2 / sr.

[0237] Although BCMA + MOLP-8-luciferase tumors gradually grew in mice receiving irrelevant scFv CAR T cells, but CAR T cells encoding 21581scFv CAR and 16747scFv CAR reduced tumor burden to background levels in all animals. The results are shown in Table 6 below.

[0238] Table 6: Average tumor size at different time points (by radiation)

[0239]

[0240]

[0241] Example 6: BCMA-specific CAR-T cells mediate cell lysis of BCMA-expressing cells

[0242] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads plus 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5 as described in Example 2 above. The transduced cells were expanded for 3 weeks with CD3 / CD28 microbeads plus 100 U / ml recombinant human IL-2 before cytolytic assays.

[0243] To determine the cytolytic capacity of chimeric antigen receptor (CAR) T cells targeting BCMA, cytolytic assays were performed using expanded CAR-T cells and multiple tumor target cell lines expressing variable 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 different ratios. Each target cell line was harvested and cultured at 2x10 6 / mL density resuspended, and then added calcein-AM dye at a concentration of 8uM for 35 minutes at 37°C. After calcein labeling, the target cells were washed twice to remove excess calcein. Subsequently, T cells and target cells were co-cultured in 96-well round-bottom plates at various ratios and cultured at 37°C for 2.5 hours when the culture supernatant was harvested. For negative controls, target cells were co-cultured with T cells produced using similar CARs designed to include irrelevant scFvs that do not recognize BCMA. As an additional CAR negative control, untransduced and amplified T cells from the same normal healthy donor were used. As a control for antigen-specific CAR-T cell-mediated killing, the chronic myeloid leukemia K562 target cell line was used because the cell line is negative for BCMA expression. In order to determine whether calcein is spontaneously released from H-929 and MOLP-8 target cell lines, each cell line was cultured in the absence of CAR-T cells. In order to determine the maximum possible release of calcein, 1% Triton was used. TM The target cell lines were cultured and lysed in Optmizer medium without X-114 detergent. In the supernatant, the relative calcein level was measured using a Viktor X4 plate reader and the percentage of cytotoxicity was calculated as ((calcein signal - spontaneous calcein release) / (calcein maximum release - spontaneous calcein release))*100.

[0244] As shown in Tables 7A-7C below, the culture composed of BCMA-targeted CAR+T cells produced using 21581 and 16747scFv induced strong cytolysis of H-929 target cells and MOLP-8 target cells. Relative to H-929 cells, it was observed that the cytotoxicity level for MOLP8 cells was low. The result is explained by the higher BCMA antigen level expressed by MOMA-8 cells expressed by H-929. For each CAR-T cell culture for BCMA, the maximum cytotoxicity for H-929 cells was observed, and the CAR-T cells for BCMA engineered using 16747scFv produced the maximum cytotoxicity for two target cell lines. When co-cultured with target cells with a ratio of a maximum of 50 T cells to a target cell, T cells and unrelated CAR-T cells (17363) that were not transduced and amplified (MOI 0) did not cause any cytolysis of MOLP-8 and H-929 target cells. This result indicates that cell lysis was observed only when the CAR structure included a BCMA-recognizing scFv (e.g., from mAb21581 and mAb16747). In addition, CAR-T cells targeting BCMA had negligible cytotoxicity against K562 cells lacking BCMA expression, indicating that BCMA expression is required to observe cell lysis.

[0245] Table 7A: BCMA-guided CAR-T cell lysis

[0246]

[0247] SD: Standard deviation

[0248] Table 7B: BCMA-guided CAR-T cell lysis

[0249]

[0250] SD: Standard deviation

[0251] Table 7C: BCMA-guided CAR-T cell lysis

[0252]

[0253] SD: Standard deviation

[0254] Example 7: In vivo cytotoxicity of CAR-T cells targeting BCMA in the bone marrow of multiple myeloma patients

[0255] CD3+ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), stimulated with CD3 / CD28 microbeads plus 100 U / ml recombinant human IL-2, and transduced with lentivirus at an MOI of 5 as described in Example 2 above. The transduced cells were expanded for 3 weeks using CD3 / CD28 microbeads plus 100 U / ml recombinant human IL-2 before cytotoxicity assays.

[0256] At harvest, the amplified CAR-T cells were washed and resuspended in complete medium (RPMI supplemented with 10% FBS, 100U / mL penicillin, 100μg / mL streptomycin and 292μg / mL L-glutamine). The bone marrow of patients with multiple myeloma was thawed and resuspended in complete medium. HS-5 stromal cells were plated in 96-well plates at 10,000 cells per well and incubated overnight. T cells and patient-derived bone marrow were added to wells containing matrix at various E: T ratios (titration of 2 times from E: T = 10: 1) and cultured at 37 ° C for 12 hours. As a CAR negative control, non-transduced and amplified T cells from the same normal healthy donor were used.

[0257] After 12 hours, flow cytometry was used to determine the survival of multiple myeloma embryonic cells. The cells were stained with a mixture of fluorophore-coupled antibodies (anti-CD4, anti-CD8, anti-CD16, anti-CD45, anti-CD90, anti-CD138 and anti-SlamF7) at 4°C in BD HorizonBrilliant Stain Buffer for 30 minutes. The cells were washed once in PBS and stained with LIVE / DEAD Fixable Dead Cell Stain at 4°C for 20-30 minutes, then washed twice in PBS and resuspended in cold Miltenyi AutoMacs Buffer. CountBright beads were added to the sample to quantify the absolute number of cells per well. Samples were analyzed on a BD FortessaX20 flow cytometer. The surviving multiple myeloma embryonic cells were selected as live single CD4- / CD8- / SlamF7+ / CD138+. The survival percentage was calculated as the absolute count of live multiple myeloma primitive cells in the treated samples normalized to live multiple myeloma cells in the untreated control.

[0258] Cultures composed of BCMA-targeted CAR+T cells produced using mAb21581 VH / VL induced robust target-specific cell lysis of multiple myeloma blasts in 2 newly diagnosed and 1 relapsed patients. At an E:T ratio of 10:1, 87-94% of multiple myeloma blasts were dissolved. Untransduced and amplified (MOI 0) T cells dissolved 34-0% of multiple myeloma blasts. The results demonstrate that BCMA-targeted CAR+T cells effectively lyse the ability of multiple myeloma blasts derived from patients in a target-specific manner. The results are shown in Table 8 below.

[0259] Table 8: Multiple myeloma blast survival % in BCMA-guided CAR-T cell lysis

[0260]

[0261] The present invention is not limited to a certain scope by the specific examples described herein. In fact, in addition to those described herein, different modifications of the present invention from the above description will be clear to those of ordinary skill in the art. Such modifications are intended to fall within the scope of the appended claims.

[0262] Some embodiments of the present invention

[0263] 1. A B cell maturation antigen (BCMA)-specific chimeric antigen receptor, which comprises, from N-terminus to C-terminus: (a) an extracellular ligand binding domain including an anti-BCMA antigen binding domain; (b) a hinge; (c) a transmembrane domain; and (d) a cytoplasmic domain including a co-stimulatory domain and a signaling domain.

[0264] 2. A chimeric antigen receptor according to embodiment 1, wherein the extracellular ligand binding domain comprises an anti-BCMA single-chain variable fragment (scFv) domain, which includes a light chain variable region (LCVR) and a heavy chain variable region (HCVR), and optionally, wherein the anti-BCMA scFv domain includes a linker between the LCVR and the HCVR.

[0265] 3. The chimeric antigen receptor of embodiment 1 or 2 further comprises a linker between the extracellular ligand binding domain and the hinge.

[0266] 4. The chimeric antigen receptor of any one of embodiments 1 to 3, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-96.

[0267] 5. The chimeric antigen receptor of any one of embodiments 1 to 4, wherein the hinge, the transmembrane domain, or both are derived from a CD8α polypeptide.

[0268] 6. A chimeric antigen receptor according to any one of embodiments 1 to 5, wherein the co-stimulatory domain comprises a 4-1BB co-stimulatory domain.

[0269] 7. A chimeric antigen receptor according to any one of embodiments 1 to 6, wherein the signaling domain comprises a CD3zeta signaling domain.

[0270] 8. The chimeric antigen receptor of any one of embodiments 1 to 7, wherein 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.

[0271] 9. The chimeric antigen receptor of embodiment 8, wherein the LCVR comprises LCDR1-LCDR2-LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 12-14-16, 28-30-32, 44-46-48, 60-62-64 or 76-78-80, respectively.

[0272] 10. The chimeric antigen receptor of any one of embodiments 1 to 9, wherein the HCVR comprises a CDR of a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, and 66.

[0273] 11. The chimeric antigen receptor of embodiment 10, wherein the HCVR comprises HCDR1-HCDR2-HCDR3 domains, which respectively comprise the amino acid sequences of SEQ ID NO: 4-6-8, 20-22-24, 36-38-40, 52-54-56 or 68-70-72.

[0274] 12. The chimeric antigen receptor of any one of embodiments 1 to 11, wherein 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.

[0275] 13. The chimeric antigen receptor of embodiment 12, wherein 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.

[0276] 14. The chimeric antigen receptor of embodiment 13, wherein the scFv domain comprises an LCVR / HCVR amino acid sequence pair comprising an amino acid sequence of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50 or 74 / 66.

[0277] 15. The chimeric antigen receptor of any one of embodiments 1 to 14, wherein the hinge comprises the amino acid sequence of SEQ ID NO: 97.

[0278] 16. The chimeric antigen receptor of any one of embodiments 1 to 15, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 98.

[0279] 17. The chimeric antigen receptor of any one of embodiments 1 to 16, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:99.

[0280] 18. The chimeric antigen receptor of any one of embodiments 1 to 17, wherein the CD3zeta signaling domain comprises the amino acid sequence of SEQ ID NO:100.

[0281] 19. The chimeric antigen receptor of embodiment 1, 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.

[0282] 20. The chimeric antigen receptor of embodiment 19, comprising the amino acid sequence of SEQ ID NO:82.

[0283] 21. The chimeric antigen receptor of embodiment 19, comprising the amino acid sequence of SEQ ID NO:84.

[0284] 22. The chimeric antigen receptor of embodiment 19, comprising the amino acid sequence of SEQ ID NO:86.

[0285] 23. The chimeric antigen receptor of embodiment 19, comprising the amino acid sequence of SEQ ID NO:88.

[0286] 24. The chimeric antigen receptor of embodiment 19, comprising the amino acid sequence of SEQ ID NO:90.

[0287] 25. An isolated nucleic acid molecule encoding the chimeric antigen receptor according to any one of embodiments 1 to 24.

[0288] 26. The nucleic acid molecule of embodiment 25, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 81, 83, 85, 87 and 89.

[0289] 27. A vector comprising the nucleic acid molecule according to embodiment 25 or 26.

[0290] 28. The vector according to embodiment 27, wherein the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

[0291] 29. The vector of embodiment 28, wherein the vector is a lentiviral vector.

[0292] 30. A cell comprising the nucleic acid molecule of embodiment 25 or 26 or the vector of any one of embodiments 27 to 29.

[0293] 31. A cell according to embodiment 30, wherein the cell is a human T cell.

[0294] 32. An engineered cell comprising a chimeric antigen receptor according to any one of embodiments 1 to 24.

[0295] 33. The engineered cell according to embodiment 32, wherein the engineered cell is an immune cell.

[0296] 34. An engineered cell according to embodiment 33, wherein the immune cell is an immune effector cell.

[0297] 35. An engineered cell according to embodiment 34, wherein the immune effector cell is a T lymphocyte.

[0298] 36. An engineered cell according to embodiment 35, wherein the T lymphocyte is an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte or a helper T lymphocyte.

[0299] 37. The engineered cell of embodiment 36, wherein the engineered cell is a CD8+ cytotoxic T lymphocyte.

[0300] 38. The engineered cell of any one of embodiments 32 to 37 for use in treating a BCMA-expressing cancer.

[0301] 39. The engineered cell of embodiment 38, wherein the BCMA-expressing cancer is multiple myeloma.

[0302] 40. An engineered human T cell comprising a chimeric antigen receptor, which comprises, from N-terminus to C-terminus: (a) an extracellular ligand binding domain comprising an anti-BCMA single-chain variable fragment (scFv) domain, which includes 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 co-stimulatory domain and a CD3zeta signaling domain.

[0303] 41. An engineered human T cell according to embodiment 40, wherein the scFv domain comprises a LCVR / HCVR amino acid sequence pair, which comprises the amino acid sequence of SEQ ID NO: 10 / 2, 26 / 18, 42 / 34, 58 / 50 or 74 / 66.

[0304] 42. An engineered human T cell according to embodiment 40 or 41, wherein the hinge comprises the amino acid sequence of SEQ IDNO:97.

[0305] 43. An engineered human T cell according to any one of embodiments 40 to 42, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:98.

[0306] 44. An engineered human T cell according to any one of embodiments 40 to 43, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:99.

[0307] 45. An engineered human T cell according to any one of embodiments 40 to 44, wherein the CD3zeta signaling domain comprises the amino acid sequence of SEQ ID NO:100.

[0308] 46. ​​The engineered human T cell of embodiment 40, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:82.

[0309] 47. The engineered human T cell of embodiment 40, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:84.

[0310] 48. The engineered human T cell of embodiment 40, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:86.

[0311] 49. The engineered human T cell of embodiment 40, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:88.

[0312] 50. The engineered human T cell of embodiment 40, comprising a chimeric antigen receptor comprising the amino acid sequence of SEQ ID NO:90.

[0313] 51. A pharmaceutical composition comprising genetically modified human T cells and a pharmaceutically acceptable carrier, wherein the genetically modified human T cells comprise a chimeric antigen receptor according to any one of embodiments 1 to 24.

[0314] 52. A pharmaceutical composition comprising the engineered cell according to any one of embodiments 32 to 37 and a pharmaceutically acceptable carrier.

[0315] 53. A pharmaceutical composition comprising the engineered cell according to any one of embodiments 40 to 50 and a pharmaceutically acceptable carrier.

[0316] 54. The pharmaceutical composition of any one of Embodiments 51 to 53, for use in treating BCMA-expressing cancer.

[0317] 55. The pharmaceutical composition of embodiment 54, wherein the BCMA-expressing cancer is multiple myeloma.

[0318] 56. Use of the chimeric antigen receptor of any one of embodiments 1 to 24, the nucleic acid molecule of embodiment 25 or 26, the vector of any one of embodiments 27 to 29, the cell of embodiment 30 or 31, or the engineered cell of any one of embodiments 32 to 37 or 40 to 50 in the preparation of a medicament for treating a BCMA-expressing cancer.

[0319] 57. The use according to embodiment 56, wherein the BCMA-expressing cancer is multiple myeloma.

[0320] 58. A method of enhancing T lymphocyte activity in a subject, comprising: introducing into the subject a T lymphocyte comprising a chimeric antigen receptor according to any one of embodiments 1 to 24.

[0321] 59. A method for treating a subject having cancer, comprising: introducing into the subject a therapeutically effective amount of T lymphocytes comprising a chimeric antigen receptor according to any one of embodiments 1 to 24.

[0322] 60. A method for stimulating a T cell-mediated immune response to 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 according to any one of embodiments 1 to 24.

[0323] 61. 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 according to any one of embodiments 1 to 24.

[0324] 62. The method of any one of embodiments 58 to 61, wherein the subject is human.

[0325] 63. The method of any one of embodiments 58 to 62, wherein the subject has multiple myeloma, B-lineage acute lymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-cell non-Hodgkin's lymphoma, leukemias and lymphomas, acute lymphocytic leukemia, Hodgkin's lymphoma, or childhood acute lymphocytic leukemia.

[0326] 64. The method of embodiment 63, wherein the subject suffers from multiple myeloma.

[0327] 65. A method of engineering a cell population to express a chimeric antigen receptor, comprising:

[0328] (a) providing an immune cell population;

[0329] (b) introducing a nucleic acid molecule encoding a chimeric antigen receptor according to any one of embodiments 1 to 24 into the immune cell;

[0330] (c) culturing the immune cell under conditions where the nucleic acid molecule is expressed; and

[0331] (d) isolating the immune cell expressing the chimeric antigen receptor on the cell surface.

[0332] 66. The method according to embodiment 65 further comprises: obtaining the immune cell population from the subject before introducing the nucleic acid molecule.

[0333] 67. A method of treating a BCMA-expressing cancer in a subject, comprising:

[0334] (a) engineering a cell population according to embodiment 66; and

[0335] (b) reintroducing the population of immune cells expressing the chimeric antigen receptor into the subject.

[0336] 68. The method of embodiment 67, wherein the BCMA-expressing cancer is multiple myeloma.

Claims

1. A B cell maturation antigen (BCMA) specific chimeric antigen receptor, which comprises from N-terminus to C-terminus: (a) an extracellular ligand binding domain comprising an anti-BCMA antigen binding domain; (b) hinges; (c) transmembrane domain; and (d) a cytoplasmic domain including a costimulatory domain and a signaling domain; Wherein, the extracellular ligand binding domain is an anti-BCMA single-chain variable fragment (scFv) domain, which includes a light chain variable region (LCVR) and a heavy chain variable region (HCVR) connected by a linker, wherein the LCVR comprises LCDR1-LCDR2-LCDR3 domains, which are amino acid sequences of SEQ ID NOs: 76-78-80, respectively; Wherein, the HCVR includes HCDR1-HCDR2-HCDR3 domains, which are the amino acid sequences of SEQ ID NO: 68-70-72 respectively.

2. The chimeric antigen receptor according to 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 according to claim 1, wherein The LCVR consists of the amino acid sequence of SEQ ID NO: 74; and the HCVR consists of the amino acid sequence of SEQ ID NO:

66.

4. The chimeric antigen receptor according to any one of claims 1 to 3, (a) wherein the hinge, the transmembrane domain, or both are derived from a CD8α polypeptide; (b) of which: The costimulatory domain comprises a 4-1BB costimulatory domain; and / or (c) wherein the signaling domain comprises a CD3zeta signaling domain.

5. The chimeric antigen receptor according to any one of claims 1 to 3, wherein: (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.

6. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:

90.

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

8. The isolated nucleic acid molecule of claim 7, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

89.

9. A vector comprising the nucleic acid molecule according to claim 7 or 8.

10. The carrier according to claim 9, wherein The vector is a DNA vector.

11. The carrier according to claim 9, wherein The vector is a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

12. An RNA vector comprising the nucleic acid molecule of claim 7.

13. The vector of claim 12, wherein the vector is a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

14. A cell comprising a nucleic acid molecule according to claim 7 or 8 or a vector according to any one of claims 9 to 13.

15. The cell according to claim 14, wherein The cells are human T cells.

16. An engineered cell comprising the chimeric antigen receptor according to any one of claims 1 to 6.

17. The engineered cell of claim 16, wherein the engineered cell is an immune cell.

18. The engineered cell of claim 16, wherein the engineered cell is an immune cell, and the immune cell is an immune effector cell.

19. The engineered cell of claim 16, wherein the engineered cell is a T lymphocyte.

20. The engineered cell of claim 16, wherein the engineered cell is a T lymphocyte, and the T lymphocyte is an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

21. The engineered cell of claim 19, which is a CD8+ cytotoxic T lymphocyte.

22. An engineered cell population, which can be obtained by: (a) providing an immune cell population obtained from a subject; (b) introducing a nucleic acid molecule encoding a chimeric antigen receptor according to any one of claims 1 to 6 into the immune cell; (c) culturing the immune cell under conditions where the nucleic acid molecule is expressed; and (d) isolating the immune cell expressing the chimeric antigen receptor on the cell surface.

23. A pharmaceutical composition comprising one of the following: (a) a genetically modified human T cell and a pharmaceutically acceptable carrier, wherein: The genetically modified human T cell comprises a chimeric antigen receptor according to any one of claims 1 to 6; or (b) the engineered cell according to claim 16 and a pharmaceutically acceptable carrier; or (c) the engineered cell according to claim 17 and a pharmaceutically acceptable carrier; or (d) the engineered cell according to claim 18 and a pharmaceutically acceptable carrier; or (e) the engineered cell according to claim 19 and a pharmaceutically acceptable carrier; or (f) the engineered cell according to claim 20 and a pharmaceutically acceptable carrier; or (g) The engineered cell according to claim 21 and a pharmaceutically acceptable carrier.

24. Use of the pharmaceutical composition according to claim 23 in the preparation of a medicament for treating multiple myeloma expressing BCMA in a subject.

25. The use according to claim 24, wherein the subject is a human individual.

26. An in vitro method of engineering a cell population to express a chimeric antigen receptor, comprising: (a) providing an immune cell population; (b) introducing a nucleic acid molecule encoding a chimeric antigen receptor according to any one of claims 1 to 6 into the immune cell; (c) culturing the immune cell under conditions where the nucleic acid molecule is expressed; as well as (d) isolating the immune cell expressing the chimeric antigen receptor on the cell surface.

27. The method of claim 26, further comprising: The immune cell population is obtained from a subject.

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