Chimeric antigen receptors targeting BCMA and methods of use thereof

Abstract

To provide anti-BCMA single-domain antibodies (sdAb), chimeric antigen receptors (CARs) comprising one or more anti-BCMA sdAbs (such as VHH fragments), engineered immune effector cells, and methods of use thereof in cancer immunotherapy.SOLUTION: The present application provides anti-BCMA single-domain antibodies (sdAb), chimeric antigen receptors (CARs) comprising one or more anti-BCMA sdAbs (such as VHH fragments), engineered immune effector cells, and methods of use thereof in cancer immunotherapy.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from International Patent Application No. PCT / CN2016 / 094408, filed August 10, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 761422000640SEQLISTING.txt, Date Recorded: August 10, 2017, Size: 520 KB).

[0003] The present invention relates to single domain antibodies, chimeric antigen receptors, and engineered immune effector cells that target BCMA, and methods of use thereof. [Background technology]

[0004] With the development of tumor immunotherapy and clinical technologies, chimeric antigen receptor T cell (CAR-T) immunotherapy is currently one of the most promising tumor immunotherapy approaches. Generally, a chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain may comprise a single-chain variable fragment (scFv) that targets a specified tumor antigen. CARs can be expressed on the surface of T cells using gene transfection technology. Upon binding to a target tumor antigen, CARs can activate T cells to mount specific anti-tumor responses in an antigen-dependent manner, without being limited by the availability of major histocompatibility complex (MHC) specific for the target tumor antigen.

[0005] Single domain antibodies (sdAbs) differ from traditional four-chain antibodies by possessing a single monomeric antibody variable domain. For example, camelids and sharks produce sdAbs, designated heavy chain-only antibodies (HcAbs), which naturally lack light chains. The antigen-binding fragment in each arm of a camelid heavy chain-only antibody is composed of a single heavy chain variable domain (V H H), which can have high affinity for antigens without the assistance of light chains. H H is the smallest known functional antigen-binding fragment with a molecular weight of approximately 15 kD.

[0006] Multiple myeloma (MM) is an incurable, aggressive plasma malignancy that is classified as a B-cell neoplasia and grows in the bone marrow in an uncontrolled manner, disrupting the normal metabolic production of blood cells and causing painful bone lesions (Garfall, AL et al., Discovery Med. 2014, 17, 37). Multiple myeloma can clinically manifest with hypercalcemia, renal failure, anemia, bone lesions, bacterial infections, hyperviscosity, and amyloidosis (Robert Z. Orlowski, Cancer Cell. 2013, 24(3)). Research and statistics show that nearly 86,000 patients are diagnosed with myeloma each year, while approximately 63,000 patients die from complications related to the disease each year (Becker, 2011). Due to the aging population, the number of myeloma cases is expected to increase year by year. Like many cancers, the cause of multiple myeloma is unknown, and there is no cure. Some treatments for multiple myeloma are similar to those for other cancers, such as chemotherapy or radiation therapy, stem cell or bone marrow transplantation, targeted therapy, or biological therapy (George, 2014). Antibody-based cellular immunotherapy is being developed to treat patients with hematological malignancies, specifically B-cell non-Hodgkin's lymphoma. Current therapies for multiple myeloma often result in remission, but nearly all patients eventually relapse. Effective immunotherapeutic agents are needed for the treatment of multiple myeloma.

[0007] LCAR-B38M, disclosed in the present invention, is a bivalent BCMA-targeted CAR-T that has already demonstrated clinical benefit in clinical trials with respect to both safety and efficacy in treating patients with refractory or relapsed multiple myeloma. In early-stage clinical trials, 33 of 35 (94%) patients had clinical remission of their multiple myeloma upon receiving LCAR-B38M CAR-T cells. Most patients experienced only mild side effects. The study was presented by the lead inventors at both the 2017 ASCO Annual Meeting (Abstract LBA3001) and a press conference (http: / / www.ascopost.com / News / 55713) that garnered extensive media coverage.

[0008] Overall, the objective response rate was 100%, with 33 patients (94%) having clear clinical remission of their myeloma (complete response, very good partial response, or partial response) within two months of receiving the CAR T cells. After following this group for more than four months, efficacy showed that 14 of 19 patients achieved stringent complete response criteria, one patient achieved a partial response, and four patients achieved very good partial response criteria.

[0009] The excellent efficacy and safety profile obtained from the LCAR-B38M clinical trial is significantly superior to several other BCMA CAR-T trials reported simultaneously at ASCO, which were widely recognized as "revolutionary breakthroughs" in the field of immunotherapy. It is noteworthy that all these BCMA CAR designs are conventional CARs in which the BCMA antigen-binding domain consists of a monovalent ScFv antibody.

[0010] The disclosures of all publications, patents, patent applications, and published patent applications referenced herein are hereby incorporated by reference in their entirety. Summary of the Invention

[0011] The present application provides an anti-BCMA single domain antibody (sdAb), one or more anti-BCMA sdAbs (V HThe present invention provides chimeric antigen receptors (CARs) comprising the H fragment, engineered immune effector cells, and methods for their use in cancer immunotherapy.

[0012] One aspect of the present application provides an anti-BCMA sdAb comprising any one of the CDR regions of SEQ ID NOs: 115 to 152. In some embodiments, the anti-BCMA sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 77, (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78, (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 79, (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 80, or (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71. (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 82; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 83; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR3 comprising the amino acid sequence of SEQ ID NO: 84; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR3 comprising the amino acid sequence of SEQ ID NO: 85. (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 10, CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and CDR3 comprising the amino acid sequence of SEQ ID NO: 86, (11) CDR1 comprising the amino acid sequence of SEQ ID NO: 11, CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and CDR3 comprising the amino acid sequence of SEQ ID NO: 87, (12) CDR1 comprising the amino acid sequence of SEQ ID NO: 12, CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and CDR3 comprising the amino acid sequence of SEQ ID NO: 88, (13) CDR1 comprising the amino acid sequence of SEQ ID NO: 13, CDR2 comprising the amino acid sequence of SEQ ID NO: 51 (14) CDR1 comprising the amino acid sequence of SEQ ID NO: 14, CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR3 comprising the amino acid sequence of SEQ ID NO: 90, (15) CDR1 comprising the amino acid sequence of SEQ ID NO: 15, CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and CDR3 comprising the amino acid sequence of SEQ ID NO: 91, (16) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and CDR3 comprising the amino acid sequence of SEQ ID NO: 92, (17) SEQ ID NO: 1 (17) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR3 comprising the amino acid sequence of SEQ ID NO: 93; (18) CDR1 comprising the amino acid sequence of SEQ ID NO: 18, CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR3 comprising the amino acid sequence of SEQ ID NO: 94; (19) CDR1 comprising the amino acid sequence of SEQ ID NO: 19, CDR2 comprising the amino acid sequence of SEQ ID NO: 57, and CDR3 comprising the amino acid sequence of SEQ ID NO: 95; (20) CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and CDR3 comprising the amino acid sequence of SEQ ID NO: (21) CDR1 comprising the amino acid sequence of SEQ ID NO: 21, CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and CDR3 comprising the amino acid sequence of SEQ ID NO: 97, (22) CDR1 comprising the amino acid sequence of SEQ ID NO: 22, CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR3 comprising the amino acid sequence of SEQ ID NO: 98, (23) CDR1 comprising the amino acid sequence of SEQ ID NO: 23, CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and CDR3 comprising the amino acid sequence of SEQ ID NO: 99, (24) CDR1 comprising the amino acid sequence of SEQ ID NO: 24,CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and CDR3 comprising the amino acid sequence of SEQ ID NO: 100; (25) CDR1 comprising the amino acid sequence of SEQ ID NO: 25, CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and CDR3 comprising the amino acid sequence of SEQ ID NO: 101; (26) CDR1 comprising the amino acid sequence of SEQ ID NO: 26, CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR3 comprising the amino acid sequence of SEQ ID NO: 102; (27) CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR3 comprising the amino acid sequence of SEQ ID NO: 103 (28) CDR1 comprising the amino acid sequence of SEQ ID NO: 28, CDR2 comprising the amino acid sequence of SEQ ID NO: 66, and CDR3 comprising the amino acid sequence of SEQ ID NO: 104, (29) CDR1 comprising the amino acid sequence of SEQ ID NO: 29, CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and CDR3 comprising the amino acid sequence of SEQ ID NO: 105, (30) CDR1 comprising the amino acid sequence of SEQ ID NO: 30, CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and CDR3 comprising the amino acid sequence of SEQ ID NO: 106, (31) CDR1 comprising the amino acid sequence of SEQ ID NO: 31, CDR2 comprising the amino acid sequence of sequence number 69, and CDR3 comprising the amino acid sequence of SEQ ID NO: 107; (32) CDR1 comprising the amino acid sequence of SEQ ID NO: 32, CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and CDR3 comprising the amino acid sequence of SEQ ID NO: 108; (33) CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and CDR3 comprising the amino acid sequence of SEQ ID NO: 109; (34) CDR1 comprising the amino acid sequence of SEQ ID NO: 34, CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and the amino acid sequence of SEQ ID NO: 110 (35) a CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a CDR2 comprising the amino acid sequence of SEQ ID NO: 73, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO: 38;CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the anti-BCMA sdAb comprises a V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. H Contains the H domain.

[0013] In some embodiments there is provided an anti-BCMA heavy chain only antibody (HCAB) or antigen binding protein comprising any one of the anti-BCMA sdAbs described above. Also provided is a BCMA epitope to which any one of the anti-BCMA sdAbs described above specifically binds to an anti-BCMA antibody (such as an anti-BCMA sdAb) that competes with any one of the anti-BCMA sdAbs described above.

[0014] In some embodiments according to any one of the anti-BCMA sdAbs described above, the anti-BCMA sdAb is a camelid antibody. In some embodiments, the anti-BCMA sdAb is a chimeric antibody. In some embodiments, the anti-BCMA sdAb is humanized. In some embodiments, the anti-BCMA sdAb is a V H It is the H fragment.

[0015] One aspect of the application provides a BCMA chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising an anti-BCMA sdAb (such as any one of the anti-BCMA sdAbs described above); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the CAR is monospecific. In some embodiments, the CAR is monovalent. In some embodiments, the CAR is multivalent (such as bivalent or trivalent). In some embodiments, the CAR is multispecific (such as bispecific). In some embodiments, the extracellular antigen binding domain comprises at least two anti-BCMA sdAbs (such as any one or more of the anti-BCMA sdAbs described above).

[0016] One aspect of the present application provides a multivalent chimeric antigen receptor (CAR) comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety and a second BCMA-binding moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, one or more of the first BCMA-binding moiety and the second BCMA-binding moiety is an anti-BCMA sdAb. In some embodiments, the first BCMA-binding moiety is a first anti-BCMA sdAb and the second BCMA-binding moiety is a second anti-BCMA sdAb. In some embodiments, the first BCMA binding moiety is an anti-BCMA sdAb and the second BCMA binding moiety is derived from a human antibody. In some embodiments, the first BCMA binding moiety is an anti-BCMA sdAb and the second BCMA binding moiety is a polypeptide ligand of BCMA. In some embodiments, the first BCMA binding moiety and the second BCMA binding moiety specifically bind to the same epitope on BCMA. In some embodiments, the first BCMA binding moiety and the second BCMA binding moiety specifically bind to different epitopes on BCMA. In some embodiments, the first BCMA binding moiety and / or the second BCMA binding moiety specifically bind to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs: 388-394. In some embodiments, the first BCMA binding moiety specifically binds to an epitope derived from SEQ ID NOs: 389 and / or 390. In some embodiments, the second BCMA binding moiety specifically binds to an epitope derived from SEQ ID NOs: 391 and / or 392.

[0017] One aspect of the present application provides a multivalent (such as bivalent or trivalent) chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising a first anti-BCMA sdAb (such as any one of the anti-BCMA sdAbs described above) and a second anti-BCMA sdAb (such as any one of the anti-BCMA sdAbs described above), (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the first The first anti-BCMA sdAb and the second anti-BCMA sdAb specifically bind to the same epitope on BCMA. In some embodiments, the first anti-BCMA sdAb and the second anti-BCMA sdAb specifically bind to different epitopes on BCMA. In some embodiments, the first anti-BCMA sdAb and / or the second anti-BCMA sdAb specifically bind to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs: 388-394. In some embodiments, the first anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NOs: 389 and / or 390. In some embodiments, the second anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NOs: 391 and / or 392.

[0018] In some embodiments according to any one of the multivalent CARs provided above, the first BCMA binding moiety (e.g., the first anti-BCMA sdAb) is located at the N-terminus of the second BCMA binding moiety (e.g., the second anti-BCMA sdAb). In some embodiments, the first BCMA binding moiety (e.g., the first anti-BCMA sdAb) is located at the C-terminus of the second BCMA binding moiety (e.g., the second anti-BCMA sdAb). In some embodiments, the first BCMA binding moiety (e.g., the first anti-BCMA sdAb) and the second BCMA binding moiety (e.g., the second anti-BCMA sdAb) are fused directly to each other via a peptide bond. In some embodiments, the first BCMA binding moiety (e.g., the first anti-BCMA sdAb) and the second BCMA binding moiety (e.g., the second anti-BCMA sdAb) are fused to each other via a peptide linker. In some embodiments, the peptide linker is about 50 or less amino acids in length (e.g., about 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 208-215.

[0019] In some embodiments according to any one of the CARs (including multivalent CARs) described above, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α or CD28. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 193 or 194.

[0020] In some embodiments according to any one of the CARs (including multivalent CARs) described above, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 197 or 198.

[0021] In some embodiments according to any one of the CARs (including multivalent CARs) described above, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 195 and / or SEQ ID NO: 196.

[0022] In some embodiments according to any one of the CARs (including multivalent CARs) described above, the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is selected from the group consisting of SEQ ID NO: 192. Contains the amino acid sequence.

[0023] In some embodiments according to any one of the CARs (including multivalent CARs) described above, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 191.

[0024] One aspect of the application provides CARs listed in Tables 4 and 5. In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-256 and 298-335.

[0025] One aspect of the present application provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152, 216-256, and 298-335.

[0026] One aspect of the present application provides an isolated nucleic acid comprising a nucleic acid sequence encoding any one of the anti-BCMA sdAbs or CARs (including multivalent CARs) described above. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 153-190, 257-297, and 336-373. In some embodiments, the isolated nucleic acid further comprises a first nucleic acid sequence encoding a first CAR, wherein a second nucleic acid sequence encoding a second CAR is operably linked to the first nucleic acid sequence via a third nucleic acid sequence encoding a self-cleaving peptide, such as a T2A, P2A, or F2A peptide. The third nucleic acid sequence is SEQ ID NO: 385. In some embodiments, the isolated nucleic acid is a DNA molecule. In some embodiments, the isolated nucleic acid is an RNA molecule.

[0027] One aspect of the present application provides a vector comprising any one of the isolated nucleic acids described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a non-viral vector.

[0028] One aspect of the present application provides an engineered immune effector cell comprising any one of the CARs (including multivalent CARs) provided above, or any one of the isolated nucleic acids described above, or any one of the vectors described above. In some embodiments, the immune effector cell is a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell. In some embodiments, the immune effector cell is a T cell.

[0029] One aspect of the present application provides a pharmaceutical composition comprising any one of the engineered immune effector cells described above and a pharmaceutically acceptable carrier. A method of treating cancer in an individual is further provided, comprising administering to the individual an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the engineered immune effector cells are autologous. In some embodiments, the engineered immune effector cells are allogeneic. In some embodiments, the cancer is a liquid cancer. In some embodiments, the cancer is multiple myeloma, acute lymphocytic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is a solid cancer such as glioblastoma. In some embodiments, the cancer is refractory or relapsed multiple myeloma.

[0030] One aspect of the present application is a combination of any one of the anti-BCMA sdAbs described above and a drug and a physiologically acceptable carrier. In some embodiments, a method of treating a disease (such as cancer) in an individual is further provided, comprising administering to the individual an effective amount of the pharmaceutical composition.

[0031] Also provided are methods of use, kits, and articles of manufacture comprising any one of the anti-BCMA sdAbs, CARs (including multivalent CARs), engineered immune effector cells, isolated nucleic acids, or vectors described above. [Brief explanation of the drawings]

[0032] [Figure 1A] Figure 1 shows the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific CARs with various anti-BCMA sdAbs against RPMI8226.Luc cells (Figure 1A), or U87MG.Luc cells (Figure 1B). [Figure 1B] Figure 1 shows the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific CARs with various anti-BCMA sdAbs against RPMI8226.Luc cells (Figure 1A), or U87MG.Luc cells (Figure 1B). [Figure 2A] Shown are the results of an in vitro cytotoxicity assay of T cells expressing an exemplary monospecific CAR with various anti-BCMA sdAbs against RPMI8226.Luc cells (Figure 2A), K562.BCMA.Luc cells (Figure 2B), or K562.CD19.Luc cells (Figure 2C). [Figure 2B] Shown are the results of an in vitro cytotoxicity assay of T cells expressing an exemplary monospecific CAR with various anti-BCMA sdAbs against RPMI8226.Luc cells (Figure 2A), K562.BCMA.Luc cells (Figure 2B), or K562.CD19.Luc cells (Figure 2C). [Figure 2C] Shown are the results of an in vitro cytotoxicity assay of T cells expressing an exemplary monospecific CAR with various anti-BCMA sdAbs against RPMI8226.Luc cells (Figure 2A), K562.BCMA.Luc cells (Figure 2B), or K562.CD19.Luc cells (Figure 2C). [Figure 3] 1 shows the results of an in vitro IFNγ release assay of T cells expressing exemplary monospecific CARs with various anti-BCMA sdAbs against K562.BCMA.Luc cells. [Figure 4A] Figure 4A shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary multivalent BCMA CAR against RPMI8226.Luc cells (Figures 4A-4B) or U87MG.Luc cells (Figure 4C). [Figure 4B] Figure 4A shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary multivalent BCMA CAR against RPMI8226.Luc cells (Figures 4A-4B) or U87MG.Luc cells (Figure 4C). [Figure 4C] Figure 4A shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary multivalent BCMA CAR against RPMI8226.Luc cells (Figures 4A-4B) or U87MG.Luc cells (Figure 4C). [Figure 5A]Figure 5 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bivalent BCMA CAR against RPMI8226.Luc cells (Figure 5A), K562.CD19.Luc cells (Figure 5B), A549.Luc cells (Figure 5C), U87MG.Luc cells (Figure 5D), or Raji.Luc cells (Figure 5E). [Figure 5B] Figure 5 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bivalent BCMA CAR against RPMI8226.Luc cells (Figure 5A), K562.CD19.Luc cells (Figure 5B), A549.Luc cells (Figure 5C), U87MG.Luc cells (Figure 5D), or Raji.Luc cells (Figure 5E). [Figure 5C] Figure 5 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bivalent BCMA CAR against RPMI8226.Luc cells (Figure 5A), K562.CD19.Luc cells (Figure 5B), A549.Luc cells (Figure 5C), U87MG.Luc cells (Figure 5D), or Raji.Luc cells (Figure 5E). [Figure 5D] Figure 5 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bivalent BCMA CAR against RPMI8226.Luc cells (Figure 5A), K562.CD19.Luc cells (Figure 5B), A549.Luc cells (Figure 5C), U87MG.Luc cells (Figure 5D), or Raji.Luc cells (Figure 5E). [Figure 5E] Figure 5 shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bivalent BCMA CAR against RPMI8226.Luc cells (Figure 5A), K562.CD19.Luc cells (Figure 5B), A549.Luc cells (Figure 5C), U87MG.Luc cells (Figure 5D), or Raji.Luc cells (Figure 5E). [Figure 5F] Figure 5F shows the results of an in vitro cytotoxicity assay of T cells expressing an exemplary bivalent BCMA CAR against K562.BCMA.Luc and K562.CD38.Luc cells. [Figure 6A]1 shows the results of an in vitro IFNγ release assay of T cells expressing an exemplary bivalent BCMA CAR on K562.BCMA.Luc cells at two different effector cell to target cell ratios. [Figure 6B] 1 shows the results of an in vitro IFNγ release assay of T cells expressing an exemplary bivalent BCMA CAR on RPMI8226.Luc, A549.Luc, K562.CD38.Luc, and Raji.Luc cells. [Figure 7A] Figures 7A-7C show the binding of three exemplary VHH fragments to K562.BCMA.Luc cells and K562.CD38.Luc cells (negative control). [Figure 7B] Figures 7A-7C show the binding of three exemplary VHH fragments to K562.BCMA.Luc cells and K562.CD38.Luc cells (negative control). [Figure 7C] Figures 7A-7C show the binding of three exemplary VHH fragments to K562.BCMA.Luc cells and K562.CD38.Luc cells (negative control). [Figure 8A] 1 shows the crystal structure of the extracellular domain of BCMA. [Figure 8B] BCMA epitope peptides are shown. [Figure 9A] Figures 9A and 9B show the results of epitope mapping assays of VHH1 and VHH2. [Figure 9B] Figures 9A and 9B show the results of epitope mapping assays of VHH1 and VHH2. [Figure 10] 1 shows the results of a competitive binding assay using CHO-BCMA cells. [Figure 11] 1 shows the in vitro cytotoxicity of donor-derived T cells expressing LCAR-B38M against RPMI8226.Luc cells. [Figure 12A] Figure 12A shows the in vitro cytotoxicity of LCAR-B38M CAR-T cells prepared from selected donors against RPMI8226.Luc cells. [Figure 12B]Figure 12B shows the in vivo antitumor activity of LCAR-B38M CAR-T cells in a tumor xenograft mouse model. Bioluminescence imaging data are shown for LCAR-B38M CAR-T-treated mice and untransduced T cell (UnT)-treated mice. [Figure 12C] Figure 12C shows the in vivo antitumor activity of LCAR-B38M CAR-T cells in a tumor xenograft mouse model. The study design and bioluminescence images of mice in the CAR-T and UnT groups are shown. [Figure 12D] Figure 12D shows the in vivo anti-tumor activity of LCAR-B38M CAR-T cells in a tumor xenograft mouse model. Images of the liver from UnT-treated mice are shown. [Figure 12E] Figure 12E shows the in vivo anti-tumor activity of LCAR-B38M CAR-T cells in a tumor xenograft mouse model. Ex vivo luciferase assay demonstrating tumors in the liver of UnT-treated mice. [Figure 13A] Figures 13A-13F show clinical parameters for two monkeys treated with LCAR-B38M CAR-T cells. Clinical parameters monitored in the study included body temperature (Figure 13A), weight (Figure 13B), complete blood count (CBC, Figures 13C and 13D), and blood chemistry and cytokine levels (Figures 13E and 13F). [Figure 13B] Figures 13A-13F show clinical parameters for two monkeys treated with LCAR-B38M CAR-T cells. Clinical parameters monitored in the study included body temperature (Figure 13A), weight (Figure 13B), complete blood count (CBC, Figures 13C and 13D), and blood chemistry and cytokine levels (Figures 13E and 13F). [Figure 13C] Figures 13A-13F show clinical parameters for two monkeys treated with LCAR-B38M CAR-T cells. Clinical parameters monitored in the study included body temperature (Figure 13A), weight (Figure 13B), complete blood count (CBC, Figures 13C and 13D), and blood chemistry and cytokine levels (Figures 13E and 13F). [Figure 13D]Figures 13A-13F show clinical parameters for two monkeys treated with LCAR-B38M CAR-T cells. Clinical parameters monitored in the study included body temperature (Figure 13A), weight (Figure 13B), complete blood count (CBC, Figures 13C and 13D), and blood chemistry and cytokine levels (Figures 13E and 13F). [Figure 13E] Figures 13A-13F show clinical parameters for two monkeys treated with LCAR-B38M CAR-T cells. Clinical parameters monitored in the study included body temperature (Figure 13A), weight (Figure 13B), complete blood count (CBC, Figures 13C and 13D), and blood chemistry and cytokine levels (Figures 13E and 13F). [Figure 13F] Figures 13A-13F show clinical parameters for two monkeys treated with LCAR-B38M CAR-T cells. Clinical parameters monitored in the study included body temperature (Figure 13A), weight (Figure 13B), complete blood count (CBC, Figures 13C and 13D), and blood chemistry and cytokine levels (Figures 13E and 13F). [Figure 14A] Figures 14A-14C show in vitro cytotoxicity assays for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells, respectively, prepared from the same three multiple myeloma patients. Figure 14A shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient A. Figure 14B shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient B. Figure 14C shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient C. [Figure 14B]Figures 14A-14C show in vitro cytotoxicity assays for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells, respectively, prepared from the same three multiple myeloma patients. Figure 14A shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient A. Figure 14B shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient B. Figure 14C shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient C. [Figure 14C] Figures 14A-14C show in vitro cytotoxicity assays for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells, respectively, prepared from the same three multiple myeloma patients. Figure 14A shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient A. Figure 14B shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient B. Figure 14C shows the in vitro cytotoxicity results for LCAR-B38M CAR-T cells and LCAR-B27S CAR-T cells prepared from multiple myeloma patient C. [Figure 15A] Figure 15A compares the structure of a VHH-based CAR with that of a conventional scFv-based CAR. The schematic structure on the left shows an exemplary monospecific monovalent CAR with an extracellular antigen-binding domain comprising a VHH domain. The schematic structure on the right shows an exemplary monospecific monovalent CAR with an extracellular antigen-binding domain comprising an scFv domain. [Figure 15B]Figure 15B compares the structure of a VHH-based CAR with two antigen-binding sites and the structure of a conventional scFv-based CAR with two antigen-binding sites. The schematic structure on the left is an exemplary CAR with an extracellular antigen-binding domain comprising two VHH domains. The two VHH domains may be the same or different. The schematic structure on the right shows an exemplary CAR with an extracellular antigen-binding domain comprising two scFv domains. The two scFv domains may be the same or different. [Figure 15C] Figure 15C shows the schematic structures of exemplary bivalent and bispecific VHH-based CARs. The schematic structure in the upper left panel shows an exemplary one-epitope, bivalent CAR with an extracellular antigen-binding domain comprising two identical VHH domains, each of which specifically binds epitope 1 of antigen A. The schematic structure in the upper right panel shows an exemplary two-epitope, bivalent CAR with an extracellular antigen-binding domain comprising a first VHH domain that specifically binds epitope 1 of antigen A and a second VHH domain that specifically binds epitope 2 of antigen A. Epitope 1 and epitope 2 of antigen A can differ in their structure and / or sequence. The schematic structure in the lower left panel shows an exemplary bispecific CAR with an extracellular antigen-binding domain comprising a first VHH domain that specifically binds antigen A and a second VHH domain that specifically binds antigen B. Antigens A and B are different antigens. [Figure 15D] Figure 15D shows the schematic structure of an exemplary VHH-based CAR with three or more VHH domains. A CAR can have multiple VHH domains fused directly to each other or via a peptide linker. The VHH domains can be the same or different. Different VHH domains can specifically bind to different epitopes on the same antigen or different antigens. [Figure 15E]Figure 15E shows exemplary engineered immune effector cells co-expressing two different VHH-based CARs. The exemplary engineered immune effector cell in the left panel co-expresses two different monospecific, monovalent CARs. The exemplary engineered immune effector cell in the middle panel co-expresses a monospecific, monovalent CAR and a bispecific or bivalent CAR. The exemplary engineered immune effector cell in the right panel co-expresses two different bispecific or bivalent CARs. The CARs may recognize different antigens. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present application provides chimeric antigen receptors (CARs) comprising an anti-BCMA single domain antibody (sdAb) and an extracellular antigen-binding domain comprising one or more BCMA-binding moieties (such as anti-BCMA sdAbs). Multivalent CARs comprising at least two binding moieties (such as sdAbs) that specifically bind to a single antigen are also provided. In some embodiments, the present application provides multivalent (such as bivalent or trivalent) CARs comprising at least two anti-BCMA sdAbs. In some embodiments, the at least two anti-BCMA sdAbs are different anti-BCMA sdAbs that specifically bind to different epitopes on BCMA. The anti-BCMA sdAbs, CARs, and engineered immune effector cells expressing the CARs described in this application are useful agents for cancer treatment.

[0034] In particular, the present application demonstrates the superior efficacy of a bivalent, two-epitope CAR (e.g., LCAR-B38M) comprising two anti-BCMA sdAbs targeting different BCMA epitopes in treating multiple myeloma in human patients in Phase I / II clinical trials. In a randomized controlled trial, 100% of patients with relapsed or refractory multiple myeloma responded to LCAR-B38M CAR-T therapy. 94% of patients achieved clear clinical remission of their myeloma within two months of receiving CAR-T therapy. Patients who achieved stringent complete response (sCR) criteria remained free of minimal residual disease for more than one year after receiving CAR-T therapy. Furthermore, LCAR-B38M CAR-T therapy was well tolerated by patients, as most experienced only mild and manageable cytokine release syndrome, a common side effect of CAR-T cell-based therapy. No patients experienced neurological side effects. In comparison, pilot clinical studies of monovalent CARs containing a single anti-BCMA sdAb demonstrated lower objective response and complete remission rates and higher relapse rates among treated patients. Prior to this application, all BCMA CARs under clinical study had only one BCMA-binding moiety in the extracellular antigen-binding domain. The improved clinical efficacy and safety of the multivalent BCMA CARs of the present application is unexpected.

[0035] Thus, one aspect of the present application provides a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a plurality of single domain antibodies (sdAbs) that specifically bind to BCMA; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0036] In another aspect, a multivalent CAR is provided comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising a first BCMA binding moiety (such as a first anti-BCMA sdAb) that specifically binds to a first epitope of BCMA, and a second BCMA binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second epitope of BCMA, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the first epitope is different from the second epitope.

[0037] Further provided are novel anti-BCMA sdAbs and CARs comprising any one or more of the anti-BCMA sdAbs described herein.

[0038] Also described herein are engineered immune effector cells (such as T cells) comprising a CAR, pharmaceutical compositions, kits, articles of manufacture, and methods of treating cancer using the engineered immune effector cells or sdAbs.

[0039] I. Definition The term "antibody" includes monoclonal antibodies (including full-length four-chain antibodies having an immunoglobulin Fc region or full-length heavy-chain-only antibodies), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab'), and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Antibodies contemplated herein include single-domain antibodies, such as heavy-chain-only antibodies.

[0040] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called a J chain, which contains 10 antigen-binding sites, whereas IgA antibodies consist of two to five basic four-chain units, which can polymerize to form multivalent aggregates with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, whereas the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain contains a variable domain (V) at its N-terminus. H ), followed by three constant domains (C for each of the α and γ chains) H ), and four C for μ and ε isotypes H Domain Each L chain has a variable domain (V L ) followed by a constant domain at the opposite end. L is VH is consistent with C L is the first constant domain of the heavy chain (C H 1) Specific amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain. H and V L and pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and chapter 6. L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. The constant domains of their heavy chains (C H Depending on the amino acid sequence of their amino acids, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are H They are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0041] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in four-chain antibodies. Camelids (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0042] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). An sdAb is capable of binding to an antigen alone, without pairing with a corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred to herein as "V H Some V H H may also be known as nanobodies. Camelid sdAbs are among the smallest known antigen-binding antibody fragments (e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nature 374:168-73 (1995)). (See also: al., Nanomedicine (Lond), 8:1013-26 (2013)). H H has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0043] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from association with all other components from its production environment. Contaminating components of its production environment, such as those resulting from recombinantly transfected cells, are materials that would typically interfere with research, diagnostic, or therapeutic uses of the antibody; these may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified to (1) greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue, or preferably silver stain. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. However, usually, an isolated polypeptide The peptide or antibody is prepared by at least one purification step.

[0044] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy and light chains, respectively, are referred to as "V H " and "V L These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site. Heavy chain-only antibodies from Camelidae species have a single heavy chain variable region, which is called a "V" H H". Therefore, V H H is V H It is a special type of

[0045] The term "variable" refers to the fact that certain segments of variable domains vary significantly in sequence among different antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy- and light-chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs of each chain are held together in close proximity by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0046] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies within the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present application may be produced by, for example, hybridoma techniques (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2002) nded. 1988), Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), phage display technology (see, for example, Clackson et al., Nature, 352:624-628 (1991), Marks et al., J. Mol. Biol. 222:581-597 (1992), Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004), Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34):1246 7-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human antibodies or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993), Jakobovits et al., Nature 362:255-258 (1993), Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0047] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0048] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, they include those having heavy and light chains, including a full-length four-chain antibody Fc region. A full-length heavy chain-only antibody is one that contains only the heavy chain (V H H, etc., and Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0049] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; single-domain antibodies (V HThese include antibodies with specificity (e.g., H), as well as multispecific antibodies formed from antibody fragments. Papain digestion of antibodies yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting the ability to readily crystallize. Fab fragments contain the entire L chain plus the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. Fab' fragments contain one or more cysteines from the antibody hinge region, and the C H F(ab')2 antibody fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of one domain. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of cations are also known.

[0050] The Fc fragment contains the carboxy termini of both heavy chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0051] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops each from the H and L chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site.

[0052] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a VFv that is linked to form a single polypeptide chain. H and V L Preferably, the sFv polypeptide is an antibody fragment containing the V H and V L The sFv further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding. For a general review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0053] "Functional fragments" of antibodies as described herein include portions of an intact antibody, generally including the antigen-binding or variable region of the intact antibody, or the Fc region of the antibody that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0054] The term "diabody" refers to a V domain that achieves inter-chain but not intra-chain V domain pairing, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H and V L Refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. H and V L Diabodies are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. See, e.g., EP 404,097, WO 93 / 11161, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0055] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include the PRIMATTZFD® antibody, in which the antigen-binding region of this antibody is purified by, for example, immunizing macaque monkeys with the antigen of interest. As used herein, "humanized antibodies" are used as a subset of "chimeric antibodies."

[0056] "Humanized" forms of non-human (e.g., camelid) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR (defined below) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions correspond to those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions which improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs typically will not exceed six in the H chain and not exceed three in the L chain. The humanized antibody also optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0057] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) are also available for the preparation of human monoclonal antibodies. van Dijk and See also van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenogeneic mice, that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies generated by human B cell hybridoma technology.

[0058] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, sdAbs comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 exhibits the highest diversity of the three HVRs and is thought to play a unique role in conferring superior specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0059] The term "complementarity-determining region" or "CDR" is used to refer to hypervariable regions as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0060] Several HVR delineations are used and encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead indicates the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. The residues of each of these HVRs are listed in Table 1 below. [Table 1]

[0061] HVRs may include the following "extended HVRs": L In the above, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and V H 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the following sequence: 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). The variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

[0062] sdAb(V H The amino acid residues of V from the Camelidae family are listed in the paper by Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240(1-2): 185-195. HAs applied to the H domain, Kabat et al. ("Sequence of proteins of immunological interest," US Public Health Services, NIH Bethesda, Md., Publication No. 91) given by V H They are numbered according to the common numbering scheme for domains. H FR1 of H contains amino acid residues 1 to 30, and V H CDR1 of H contains amino acid residues 31 to 35, and CDR1 of V H FR2 of H contains amino acid residues 36 to 49, and V H CDR2 of H contains amino acid residues 50 to 65, and V H FR3 of H contains amino acid residues 66 to 94, and V H CDR3 of H contains amino acid residues 95 to 102, and V H FR4 of H comprises amino acid residues 103 to 113. In this regard, as is well known in the art, H About Domains and V H It should be noted that for H domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering).

[0063] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for heavy or light chain variable domains in the organization of antibodies in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a given antibody by matching the antibody's sequence with the "standard" Kabat-numbered sequence at the regions of homology.

[0064] Unless otherwise indicated herein, the numbering of residues in immunoglobulin heavy chains is that of the EU index as in Kabat et al., supra. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0065] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0066] "Human consensus framework" or "acceptor human framework" refers to the human immunoglobulin V L or V H A framework representing the most commonly occurring amino acid residues in a framework sequence selection. Generally, human immunoglobulin V L or V H The selection of sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is selected from the set of sequences described in Kabat et al., Sequences of Proteins of the Invention. f Immunological Interest,5 thPublic Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include V L The subgroups can be subgroup kappa I, kappa II, kappa III, or kappa IV as in Kabat et al., supra. Additionally, for VH, the subgroups can be subgroup I, subgroup II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above, such as when human framework residues are selected based on their homology to the donor framework by aligning the donor framework sequence with a collection of different human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence as the donor framework, or it can contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer.

[0067] For example, an "amino acid modification" at a specified position in the Fc region refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a specified residue means an insertion within one to two residues thereof. The insertion may be on the N-terminal or C-terminal side of the specified residue. A preferred amino acid modification herein is a substitution.

[0068] An "affinity matured" antibody is one that has one or more changes in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen compared to a parent antibody that does not possess those change(s). In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity matured antibodies. H and V L Affinity maturation by domain shuffling (random mutagenesis of HVR and / or framework residues) has been described, for example, in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994), Schier et al. Gene 169:147-155 (1995), Yelton et al. J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0069] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as binding, between a target and an antigen-binding protein (such as a CAR or sdAb), which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antigen-binding protein (such as a CAR or sdAb) that specifically binds to a target (which may be an epitope) is an antigen-binding protein (such as a CAR or sdAb) that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In some embodiments, the extent to which an antigen-binding protein (such as a CAR or sdAb) binds to an unrelated target is less than about 10% of that of the antigen-binding protein (such as a CAR or sdAb), as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antigen binding protein (such as a CAR or sdAb) that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. ... ) specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding.

[0070] The term "specificity" refers to the selective recognition of an antigen-binding protein (such as a CAR or sdAb) for a specific epitope of an antigen. For example, natural antibodies are monospecific. The term "multispecificity" as used herein indicates that an antigen-binding protein (such as a CAR or sdAb) has two or more antigen-binding sites, at least two of which bind to different antigens. "Bispecificity" as used herein indicates that an antigen-binding protein (such as a CAR or sdAb) has two different antigen-binding specificities. The term "monospecific" CAR as used herein refers to an antigen-binding protein (such as a CAR or sdAb) that has one or more binding sites, each of which binds to the same antigen.

[0071] The term "valent," as used herein, refers to the presence of a specified number of binding sites in an antigen-binding protein (such as a CAR or sdAb). For example, a natural antibody, or a full-length antibody, has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites, respectively, in an antigen-binding protein (such as a CAR or sdAb).

[0072] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (either a native-sequence Fc region or an amino acid sequence variant Fc region) and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. A "reduced or minimized" antibody effector function indicates a reduction of at least 50% (alternatively, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) from that of a wild-type or unmodified antibody. Antibody effector functions are readily determinable and measurable by those skilled in the art. In preferred embodiments, the antibody effector functions of complement fixation, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity are affected. In some embodiments, effector function is eliminated through a mutation in the constant region that eliminates glycosylation, e.g., an "effectorless mutation." In one aspect, the effectorless mutation is HThe N297A or DANA mutation (D265A+N297A) in the 2-region is shown in Figure 1. Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Additional mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques such as expression in non-glycosylated host cells (e.g., E. coli) or host cells that result in altered glycosylation patterns that are ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0073] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. Antibodies are required to "arm" the cytotoxic cells and kill the target cells by this mechanism. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRII. γRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0074] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, intact antibody compositions may include antibody populations with all K447 residues removed, antibody populations lacking the removed K447 residue, and antibody populations having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0075] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or CAR) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen, or a CAR and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present application. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0076] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

[0077] With respect to peptide, polypeptide, or antibody sequences, "percent (%) amino acid sequence identity" and "homology" are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0078] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to graft one or more antigen specificities onto immune effector cells, such as T cells. Some CARs are also known as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises an extracellular antigen-binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T" refers to a T cell expressing a CAR. "BCMA CAR" refers to a CAR with an extracellular antigen-binding domain specific for BCMA. "Dual epitope CAR" refers to a CAR with extracellular binding domains specific for two different epitopes on BCMA.

[0079] An "isolated" nucleic acid molecule encoding a CAR or sdAb described herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, an isolated nucleic acid is free from association with all components associated with the production environment. An isolated nucleic acid molecule encoding a polypeptide or antibody of the present invention is in a form other than the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid encoding a polypeptide or antibody of the present invention that naturally exists in a cell.

[0080] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0081] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0082] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0083] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is subsequently reintroduced into the individual.

[0084] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0085] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one in which exogenous nucleic acid has been transferred or introduced into a host cell. A cell that has been transfected, transformed, or transduced with an acid. This cell includes the primary subject cell and its progeny.

[0086] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, "transfectants" and "transfected cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

[0087] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0088] As used herein, "treatment" or "treating" refers to an approach to obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include one or more of the following: alleviating one or more symptoms attributable to the disease, attenuating the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the condition of the patient, providing remission (partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving quality of life, and / or extending survival. "Treatment" also encompasses alleviation of the pathological consequences of cancer. The methods of the present application contemplate any one or more of these therapeutic aspects.

[0089] As used herein, "individual" or "subject" refers to a mammal, including, but not limited to, a human, cow, horse, cat, dog, rodent, or primate. In some embodiments, the individual is a human.

[0090] As used herein, the term "effective amount" refers to an amount of an agent, e.g., an sdAb, engineered immune effector cell, or pharmaceutical composition thereof, sufficient to treat a particular disorder, condition, or disease, e.g., to ameliorate, alleviate, relieve, and / or delay one or more of its symptoms. With respect to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce the rate of tumor growth (e.g., suppress tumor growth), or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. An effective amount of the drug or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) prevent, slow, delay to some extent, and preferably stop cancer cell invasion into peripheral organs, (iv) prevent (i.e., slow to some extent, and preferably stop) tumor metastasis, (v) prevent tumor growth, (vi) prevent or delay tumor onset and / or recurrence, and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer.

[0091] "Subsidized status" refers to an individual who has a history of cancer and is generally (but not necessarily) receiving "Adjuvant" refers to the clinical situation in which an individual has responded to therapy, including, but not limited to, surgery (e.g., resection), radiation therapy, and chemotherapy. However, because of the individual's history of cancer, these individuals are considered at risk for developing the disease. Treatment or administration in the "adjuvant setting" refers to subsequent treatment modalities. The degree of risk (e.g., if an individual in the adjuvant setting is considered "high risk" or "low risk") depends on several factors, most commonly the extent of the disease when first treated.

[0092] "Neoadjuvant setting" refers to the clinical setting in which the method is administered before primary / causal therapy.

[0093] As used herein, "delaying" the onset of cancer means suspending, preventing, delaying, slowing, stabilizing, and / or postponing the onset of the disease. This delay can be of varying duration depending on the disease history and / or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method for "delaying" the onset of cancer is one that reduces the likelihood of disease onset within a given time frame and / or reduces the extent of disease within a given time frame compared to when the method is not used. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. Cancer onset may be detectable using standard methods, including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset also refers to cancer progression, which may be undetectable initially, and includes onset, recurrence, and onset.

[0094] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such a formulation is sterile. A "sterile" formulation is aseptic or free of all viable microorganisms and their spores.

[0095] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Often, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers. amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™, or polyethylene glycol (PEG).

[0096] A contemplated "diluent," as used herein, is one that is pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful in the preparation of liquid formulations, e.g., formulations to be reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. In an alternative embodiment, the diluent is an aqueous salt solution and / or or a buffer.

[0097] A "preservative" is a compound that can be added to the formulation herein to reduce bacterial activity. The addition of a preservative can, for example, facilitate the production of a multi-use (multi-dose) formulation. Examples of possible preservatives include, for example, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

[0098] A "stable" formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For rapid screening, a formulation can be stored at 40°C for 2 weeks to 1 month, at which point stability is measured. If the formulation is stored at 2-8°C, it should generally be stable at 30°C or 40°C for at least 1 month and / or at 2-8°C for at least 2 years. If a formulation is stored at 30°C, generally the formulation should be stable at 30°C for at least 2 years and / or stable at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation can be one in which less than about 10%, preferably less than about 5%, of the protein is present as aggregates in the formulation. In other embodiments, any increase in aggregate formation during storage of the formulation can be determined.

[0099] A "reconstituted" formulation is one prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed throughout. The reconstituted formulation is suitable for administration (e.g., subcutaneous administration) to a patient treated with the protein of interest, and in some embodiments of the present application may be suitable for parenteral or intravenous administration.

[0100] An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250-350 mOsm. The term "hypotonic" describes a formulation that has an osmotic pressure lower than that of human blood. Similarly, the term "hypertonic" is used to describe a formulation that has an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using a vapor pressure or ice osmometer. The formulations of the present invention are hypertonic as a result of the addition of salts and / or buffers.

[0101] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0102] Reference herein to "about" a value or parameter includes (and describes) variations on that value or parameter itself. For example, a statement referring to "about X" includes a statement of "X."

[0103] As used herein, reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, a method for treating cancer of type X is not used means that a method for treating cancers of types other than X is used.

[0104] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0105] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0106] II. Anti-BCMA Single Domain Antibodies One aspect of the present application provides isolated single domain antibodies (referred to herein as "anti-BCMA sdAbs") that specifically bind to BCMA, such as human BCMA. In some embodiments, the anti-BCMA sdAb modulates BCMA activity. In some embodiments, the anti-BCMA sdAb is an antagonist antibody. The anti-BCMA antibodies described herein Further provided are antigen binding fragments derived from any one of the sdAbs, and antigen binding proteins comprising any one of the anti-BCMA sdAbs described herein. Exemplary anti-BCMA sdAbs are listed in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0107] B-cell maturation antigen (BCMA) (also known as CD269) is a member of the tumor necrosis factor receptor superfamily, i.e., TNFRSF17 (Thompson et al., J. Exp. Medicine, 192(1):129-135, 2000). Human BCMA is expressed almost exclusively on plasma cells and multiple myeloma cells (e.g., Novak et al., Blood, 103(2):689-694, 2004; Neri et al., Clinical Cancer Research, 73(19):5903-5909; Felix et al., Mol. Oncology, 9(7):1348-58, 2015). BCMA can bind to B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (e.g., Mackay et al., 2003 and Kalled et al., Immunological Review, 204:43-54, 2005). BCMA may be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma. High-affinity antibodies can block the binding between BCMA and its natural ligands BAFF and APRIL. Anti-BCMA sdAbs can be used in combination with cellular immunotherapy using CAR-T cells, for example, to enhance the cytotoxic effect on tumor cells.

[0108] In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 115. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 116. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 117. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 118. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 119. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 120. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 121. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 122. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 123. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 124. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 125. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 126. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 127. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 128. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 129.In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 130. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 131. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 132. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 133. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 134. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 135. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 136. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 137. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 138. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 139. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 140. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 141. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 142. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 143. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 144. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 145. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 146. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 147. In some embodiments there is provided an anti-BCMA sdAb which comprises one, two or all three CDRs of the amino acid sequence of SEQ ID NO: 148. In some embodiments there is provided an anti-BCMA sdAb which comprises one, two or all three CDRs of the amino acid sequence of SEQ ID NO: 149.In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 150. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 151. In some embodiments, an anti-BCMA sdAb is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 152. In some embodiments, the anti-BCMA sdAb is camelid. In some embodiments, the anti-BCMA sdAb is humanized. In some embodiments, the anti-BCMA sdAb comprises an acceptor human framework, for example, a human immunoglobulin framework or a human consensus framework.

[0109] In some embodiments, an anti-BCMA sdAb is provided that comprises at least one, at least two, or all three CDRs selected from: (a) CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 1-38, (b) CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 39-76, and (c) CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 77-114. In some embodiments, the anti-BCMA sdAb is camelid. In some embodiments, the anti-BCMA sdAb is humanized. In some embodiments, the anti-BCMA sdAb comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0110] In some embodiments, the CDR1 comprises: (a) a CDR1 having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-38; (b) a CDR2 having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 39-76; and (c) a CDR2 having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 77-114.

[0023] Anti-BCMA sdAbs are provided that comprise three CDRs, including a CDR3 that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference amino acid sequence. In some embodiments, a CDR that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-BCMA sdAb comprising that sequence retains the ability to bind BCMA. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs: (a) a CDR1 having about one, two, three, or four amino acid substitutions (e.g., conservative substitutions), insertions, or deletions relative to an amino acid sequence selected from SEQ ID NOs: 1-38; (b) a CDR2 having about one, two, three, or four amino acid substitutions (e.g., conservative substitutions), insertions, or deletions relative to an amino acid sequence selected from SEQ ID NOs: 39-76; and (c) a CDR3 having about one, two, three, or four amino acid substitutions (e.g., conservative substitutions), insertions, or deletions relative to an amino acid sequence selected from SEQ ID NOs: 77-114. In some embodiments, the anti-BCMA sdAb is affinity matured. In some embodiments, the anti-BCMA sdAb is camelid. In some embodiments, the anti-BCMA sdAb is humanized. In some embodiments, the anti-BCMA sdAb comprises a receptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0111] In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 5, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 81. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, there is provided an anti-BCMA sdAb that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 83. In some embodiments, there is provided an anti-BCMA sdAb that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 84.In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, (a) SEQ ID NO: Anti-BCMA sdAbs are provided that comprise three CDRs, including (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments, anti-BCMA sdAbs are provided that comprise three CDRs, including (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 88. sdAbs are provided. In some embodiments, anti-BCMA sdAbs are provided that comprise three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, anti-BCMA sdAbs are provided that comprise three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, anti-BCMA sdAbs are provided that comprise three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 16, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 19, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 57, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 96. sdAbs are provided. In some embodiments, anti-BCMA sdAbs are provided that comprise three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, anti-BCMA sdAbs are provided that comprise three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 22, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, anti-BCMA sdAbs are provided that comprise three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 24, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 100. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, including (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 27, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 28, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 66. and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 30, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 31, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 107. In some embodiments, an anti-BCMA sdAb is provided which comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 108. In some embodiments, an anti-BCMA sdAb is provided which comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 33, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 109. In some embodiments, an anti-BCMA sdAb is provided which comprises three CDRs, comprising (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 34, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 110. In some embodiments, there is provided an anti-BCMA sdAb that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 35, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 73, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 111. In some embodiments, there is provided an anti-BCMA sdAb that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 36, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 112.In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, an anti-BCMA sdAb is provided that comprises three CDRs, comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO: 38, (b) a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the anti-BCMA sdAb is camelid. In some embodiments, the anti-BCMA sdAb is humanized. In some embodiments, the anti-BCMA sdAb comprises a receptor human framework, for example a human immunoglobulin framework or a human consensus framework.

[0112] In some embodiments, an anti-BCMA sdAb (i.e., an anti-BCMA sdAb comprising a particular CDR1, CDR2, and / or CDR3), including any of the embodiments described above, comprises a V having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 115-152. H In some embodiments, the VH domain has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the VH domains. HThe H sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-BCMA sdAb comprising that sequence retains the ability to bind to BCMA. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted by an amino acid sequence selected from SEQ ID NOs: 115-152. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., within the FRs). Optionally, the anti-BCMA sdAb comprises an amino acid sequence selected from SEQ ID NOs: 115-152, including post-translational modifications of that sequence.

[0113] In some embodiments, a V has an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. H An isolated anti-BCMA sdAb is provided that comprises an H domain. In some embodiments, a polypeptide is provided having an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152.

[0114] In some embodiments, functional epitopes can be mapped by combinatorial alanine scanning. In this process, a combinatorial alanine scanning strategy can be used to identify amino acids in the BCMA protein required for interaction with an anti-BCMA sdAb. In some embodiments, the epitope is conformational, and a crystal structure of an anti-BCMA sdAb bound to BCMA can be used to identify the epitope. In some embodiments, the present application provides an epitope of BCMA derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-394. In some embodiments, the present application provides an epitope of BCMA comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-394.

[0115] In some embodiments, the present application provides antibodies that compete with any one of the anti-BCMA sdAbs described herein for binding to BCMA. In some embodiments, the present invention provides antibodies that compete with the anti-BCMA sdAbs provided herein for binding to an epitope on BCMA. In some embodiments, antibodies are provided that bind to the same epitope as an anti-BCMA sdAb comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, antibodies are provided that specifically bind to BCMA in competition with an anti-BCMA sdAb comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152.

[0116] In some embodiments, a competition assay can be used to identify monoclonal antibodies that compete with the anti-BCMA sdAbs described herein for binding to BCMA. Competition assays can be used to determine whether two antibodies bind to the same epitope by recognizing the same or sterically overlapping epitope, or by one antibody competitively inhibiting the binding of another antibody to the antigen. In certain embodiments, such competing antibodies bind to the same epitope bound by the antibodies described herein (e.g., a BCMA epitope derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-394). Exemplary competition assays include, but are not limited to, routine assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for mapping antibody binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, two antibodies are said to bind to the same epitope if each blocks the binding of the other by 50% or more. In some embodiments, antibodies that compete with anti-BCMA sdAbs described herein are camelid, chimeric, humanized, or human antibodies. In some embodiments, the present application provides antibodies that compete with camelid, chimeric, humanized, or human anti-BCMA sdAbs described herein.

[0117] In some embodiments, an anti-BCMA antibody or antigen binding protein binding comprising any one of the anti-BCMA sdAbs described above is provided. In some embodiments, the anti-BCMA antibody is a monoclonal antibody, including a camelid, chimeric, humanized, or human antibody. In some embodiments, the anti-BCMA antibody is an antibody fragment, e.g., Ba, V H In some embodiments, the anti-BCMA antibody is a full-length heavy chain-only antibody comprising an Fc region of any antibody class or isotype, such as IgG1 or IgG4. In some embodiments, the Fc region has reduced or minimized effector function.

[0118] In some embodiments, an anti-BCMA antibody (such as an anti-BCMA sdAb) or antigen binding protein according to any of the above embodiments may incorporate any of the features described in "Antibody Features" sections 1-7 below, either alone or in combination.

[0119] In some embodiments, an isolated nucleic acid encoding any one of the anti-BCMA antibodies (e.g., anti-BCMA sdAbs) described above is provided. In some embodiments, an isolated nucleic acid encoding an anti-BCMA sdAb is provided, the nucleic acid comprising a sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 153-190. In some embodiments, an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 153-190 is provided. In some embodiments, a vector (e.g., an expression vector) comprising such a nucleic acid is provided. In some embodiments, a host cell comprising such a nucleic acid is provided. In some embodiments, methods of making an anti-BCMA antibody are provided, the methods comprising culturing a host cell comprising nucleic acid encoding an anti-BCMA antibody as provided above under conditions suitable for expression of the anti-BCMA antibody, and optionally recovering the anti-BCMA antibody from the host cell (or host cell culture medium).

[0120] Antibody Characteristics 1. Antibody affinity In some embodiments, the anti-BCMA antibodies provided herein have a concentration of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0121] In some embodiments, the Kd is determined by measuring the Kd of the Fab or V version of the antibody of interest, as illustrated by the following assay: H The solution binding affinity of a Fab to an antibody is measured by radiolabeled antigen binding assay (RIA) performed using the H fragment and its antigen. For example, the solution binding affinity of a Fab to an antibody is measured by binding the Fab to the lowest concentration ( 125 I) It is measured by equilibrating with labeled antigen and then capturing the bound antibody with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)).

[0122] In some embodiments, Kd is measured using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip of approximately 10 response units (RU) at 25°C. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, the Fab or V of the antibody of interest is used.H Two-fold serial dilutions of H (0.78 nM to 500 nM) were incubated in 0.05% polysorbate 20 (TWEEN 1000) at 25 °C. The assay is performed in PBS with PBST (-20™) surfactant at a flow rate of approximately 25 μL / min. The association rate (k) and dissociation rate (k) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (K) is calculated as the ratio of k / k. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate is 10 k / k, as determined by the surface plasmon resonance assay described above, the assay is performed. 6 M -1 s -1 If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen as measured in a spectrometer such as a spectrophotometer equipped with stopped-flow (Aviv Instruments) or an 8000 Series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0123] 2. Antibody fragments In some embodiments, the antibodies provided herein are antibody fragments, including Fab, Fab', Fab'-SH, F(ab'), Fv, and scFv fragments, V HExamples of antibody fragments include, but are not limited to, Fab and F(ab')2 fragments, as well as other fragments described below. A review of certain antibody fragments is also provided in Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFvs, see, e.g., Pluckthun, in *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), and also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046.

[0124] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, and Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0125] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0126] 3. Chimeric and humanized antibodies In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567 and Morrison et al. al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a camelid species such as a llama) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0127] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, a humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0128] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are also described, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface reconstitution"), Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0129] Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0130] In some embodiments, the sdAb is modified, e.g., humanized, to reduce its immunogenicity to heterologous species while not reducing the domain's native affinity for the antigen. For example, the antibody variable domain (V) of a llama antibody is modified, e.g., humanized, to reduce its immunogenicity to heterologous species while not reducing the domain's native affinity for the antigen. H H) can be determined, and one or more of the camelid amino acids in the framework regions are replaced with their human counterparts as found in the human consensus sequence, such that the polypeptide does not lose its typical characteristics, i.e., the humanization does not significantly affect the antigen-binding capacity of the resulting polypeptide. Humanization of camelid sdAbs requires the introduction and mutagenesis of a limited number of amino acids within a single polypeptide chain. This is in contrast to humanization of scFv, Fab', (Fab')2, and IgG, which requires the introduction of amino acid changes in two chains (light and heavy) and the preservation of the assembly of both chains.

[0131] V H Single domain antibodies comprising an H domain can be humanized to have human-like sequences. In some embodiments, the V domains used herein H The FR region of the H domain is H Humanized V comprises any one of at least about 50%, 60%, 70%, 80%, 90%, 95% or more amino acid sequence homology to the framework regions. H One exemplary class of H domain is the V H H is characterized in that it possesses an amino acid at position 45 (e.g., L45) from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine according to the Kabat numbering system, and tryptophan at position 103. Thus, polypeptides belonging to this class are characterized in that they possess ... H Showing high amino acid sequence homology to the framework regions, the polypeptides can be directly administered to humans without expecting an undesired immune response therefrom and without the burden of further humanization.

[0132] Another exemplary class of humanized camelid sdAbs is described in WO03 / 035694 and includes V sequences typically found in conventional antibodies of human origin or from other species, but derived from double-chain antibodies. H The peptides belonging to these two classes contain hydrophobic FR2 residues that compensate for this loss of hydrophilicity by substituting the conserved tryptophan residue present in human V with a charged arginine residue at position 103. Thus, peptides belonging to these two classes are highly complementary to human V H Showing high amino acid sequence homology to the framework regions, the peptides can be directly administered to humans without the burden of further humanization, without expecting any undesired immune response therefrom.

[0133] 4. Human antibodies In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Transgenic mice or rats capable of producing fully human sdAbs are known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0134] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is extrachromosomally present or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describing XENOMOUSE™ technology), U.S. Patent No. 5,770,429 (describing HUMAB® technology), U.S. Patent No. 7,041,870 (describing KM MOUSE® technology), and U.S. Patent Application Publication No. US2007 / 0061900 (describing VELOCIMOUSE® technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0135] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described by Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185:91 (2005).

[0136] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0137] V directed against a specific antigen or targetH One technique for obtaining H sequences is to suitably immunize a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies directed against the antigen or target) and to immunize the V H obtaining a suitable biological sample (such as a blood sample, serum sample or B cell sample) from said transgenic mammal containing (a nucleic acid sequence encoding) an H sequence, and then, starting from said sample, generating a V directed against said antigen or target using any suitable technique known per se (such as any of the methods described herein or hybridoma techniques); H The heavy chain antibody-expressing mouse involves generating a heavy chain antibody sequence. For example, for this purpose, the heavy chain antibody-expressing mouse and additional methods and techniques described in WO02 / 085945, WO04 / 049794, and WO06 / 008548, and Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10;103(41):15130-5, can be used. For example, such a heavy chain antibody-expressing mouse can express a heavy chain antibody having any suitable (single) variable domain, such as a (single) variable domain derived from a natural source (e.g., a human (single) variable domain, a camelid (single) variable domain, or a shark (single) variable domain), as well as a synthetic or semi-synthetic (single) variable domain.

[0138] 5. Library-derived Antibodies Antibodies of the present application can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and see, for example, McCafferty et al., Nature 348:552-554, Clackson et al. al.,Nature 352:624-628(1991), Marks et al.,J.Mol.Biol.222:581-597(1992), Marks and Bradbury,in Methods in Molecular Biology 248:161-175(Lo,ed.,Human Press,Totowa,NJ,2003),Sidhu et al. al., J.Mol.Biol.338(2):299-310(2004), Lee et al.,J.Mol.Biol.340(5):1073-1093(2004), Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004); and Lee et al. al.,J.Immunol.Methods 284(1-2):119-132 (2004). Methods for constructing sdAb libraries are described, see, for example, U.S. Patent No. 7,371,849.

[0139] In certain phage display methods, V H and V LGene repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunized sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide single-source antibodies against a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J., 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0140] Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

[0141] 6. Multispecific antibodies In some embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different sites. In some embodiments, one binding specificity is for an antigen selected from the group consisting of CD19, CD20, BCMA, and CD38, and the other is for any other antigen. In some embodiments, bispecific antibodies can bind to two different epitopes of an antigen selected from the group consisting of CD19, CD20, BCMA, and CD38. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing an antigen selected from the group consisting of CD19, CD20, BCMA, and CD38.

[0142] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for producing multispecific antibodies include combining two immunoglobulins with different specificities. Recombinant co-expression of heavy chain-light chain pairs (Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBOJ. 10:3655 (1991)), and "knob-in-hole" manipulation (see, for example, US Pat. No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 148(5):1547-1553 (1992)). al., J. Immunol., 152:5368 (1994)), as well as by preparing trispecific antibodies (e.g., Tutt et al. J. Immunol. 147:60 (1991), and by generating polypeptides that include tandem single domain antibodies (see, e.g., U.S. Patent Application No. 20110028695, and Conrath et al. J. Biol. Chem., 2001;276(10):7346-50). Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, e.g., US2006 / 0025576A1).

[0143] 7. Antibody variants In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0144] a) Substitution, insertion, and deletion variants In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 3 under the heading "Preferred Substitutions." More substantial changes are provided in Table 3 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 3]

[0145] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basic: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe.

[0146] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0147] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have modified (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. Exemplary substitutional variants include, for example, These are affinity matured antibodies, which can be conveniently generated using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0148] Modifications (e.g., substitutions) may be made within HVRs, for example, to improve antibody affinity. Such modifications may be made to HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or to SDRs (a-CDRs), resulting in mutant V H or V Lare tested for binding affinity. Affinity maturation by construction of and reselection from a secondary library is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. Often, CDR-H3 and CDR-L3 are specifically targeted.

[0149] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within an HVR. Such modifications may be outside an HVR "hot spot" or CDR. The variant V provided above H In some embodiments of the H sequence, each HVR is either unaltered or has no more than one, two, or three amino acid substitutions.

[0150] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" and is described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted as candidates for substitution or can be eliminated. The mutants can be screened to determine whether they have the desired properties.

[0151] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0152] b) Glycosylation variants In some embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0153] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present application may be made to generate antibody variants with certain improved properties.

[0154] In some embodiments, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.) and US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, and US2004 / 013214. 0, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249(2004), Yamane-Ohnuki et al. al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1 (Presta, L, and WO2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0155] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0156] c) Fc region variants In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0157] In some embodiments, the present application contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for uses in which the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express Fc(RIII) only, whereas monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7063-7064 (1986)). 82:1499-1502 (1985), 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be measured in vivo, e.g., by assaying a target molecule in vivo using a method such as that described by Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and thereby lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738). -2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0158] Antibodies with reduced effector function include antibodies with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, such as the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0159] Certain antibody variants have been described with improved or reduced binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0160] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0161] In some embodiments, modifications made in the Fc region result in altered (i.e., either improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0162] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0163] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0164] d) Cysteine ​​Engineered Antibody Variants In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMabs," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, e.g., drug moieties or linker-drug moieties, to generate immunoconjugates as further described herein. In some embodiments, any of the following residues may be selected from the group consisting of: Any one or more of: A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region may be substituted with cysteine. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0165] e) Antibody derivative In some embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0166] In some embodiments, conjugates of an antibody and a nonproteinaceous moiety are provided that can be selectively heated by exposure to radiation. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including, but not limited to, wavelengths that heat the nonproteinaceous moiety to temperatures that do not harm normal cells but that kill cells proximal to the antibody-nonproteinaceous moiety.

[0167] Preparation method The antibodies described herein (such as sdAbs) may be prepared using any method known in the art or described herein.

[0168] Methods for preparing sdAbs have been described. See, for example, Els Pardon et al, Nature Protocols, 2014;9(3):674. Single domain antibodies (V H H, etc.) can be obtained using methods known in the art, for example by immunising a Camelidae species (such as a camel or llama) and obtaining hybridomas therefrom, or by cloning a library of sdAbs using molecular biology techniques known in the art and then selecting individual clones of the unselected library by ELISA or using phage display.

[0169] For recombinant production of sdAbs, nucleic acid encoding the sdAb is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the sdAb is readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains). Many vectors are available. The choice of vector will depend in part on the host cell to be used. Generally, preferred host cells are prokaryotic They are either of biological or eukaryotic (generally mammalian) origin.

[0170] 1. Polyclonal antibodies Polyclonal antibodies are generally raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R 1 N=C=NR, where R and R 1 It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using the following groups (wherein are independently lower alkyl groups): (A, B, C, C, D, E, F, G, G, G, H, G, I, M ...

[0171] Animals are immunized against the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer reaches a plateau. Conjugates can also be produced in recombinant cell culture as protein fusions. Agglutinating agents such as alum are also suitable for enhancing the immune response.

[0172] 2. Monoclonal antibodies Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0173] For example, monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0174] In the hybridoma method, a mouse, or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles of Immunotherapy, Vol. 1, No. 1, pp. 111-114, 1997). and Practice, pp.59-103 (Academic Press, 1986).

[0175] The immunizing agent typically includes an antigen protein or a fusion variant thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoc lonal Antibodies: Principles and Practice, Academic Press (1986), pp.59-103.

[0176] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that inhibit the growth of HGPRT-deficient cells.

[0177] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these are those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and American Type I myeloma cells. Preferred are mouse myeloma lines, such as SP-2 cells (and their derivatives, e.g., X63-Ag8-653), available from the Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0178] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0179] The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0180] After hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as tumors in a mammal.

[0181] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0182] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 and above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to synthesize the monoclonal antibody in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Rev. 130:151-188 (1992).

[0183] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0184] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0185] The monoclonal antibodies described herein can be monovalent, and their preparation is known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at a point within the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residue can be substituted with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments, specifically Fab fragments, can be accomplished using routine techniques known in the art.

[0186] Chimeric or hybrid antibodies also can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate. (mercaptobutyrimidate).

[0187] 3. Recombinant production in prokaryotic cells a) Vector construction Polynucleotide sequences encoding the antibodies of the present application can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors are available and known in the art and can be used in the present invention. Selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0188] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used in connection with these hosts. The vector usually carries a replication site and marking sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used for the expression of specific antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

[0189] Additionally, phage vectors containing replicon and control sequences compatible with host microorganisms can be used as transforming vectors in connection with these hosts. For example, bacteriophages such as GEM™-11 can be utilized to generate recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0190] The expression vector of the present application may contain two or more promoter-cistron pairs, one encoding each of the polypeptide components. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes: inducible promoters and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription of the cistron under their control in response to changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

[0191] Numerous promoters recognized by a variety of potential host cells are well known. The selected promoter may be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and / or expression of the target gene. In some embodiments, a heterologous promoter is utilized because heterologous promoters generally result in greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0192] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the -lactamase and lactose promoter system, the tryptophan (trp) promoter system, and hybrid promoters, such as the tac or trc promoter. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences have been published, allowing one skilled in the art to operably link them to cistrons encoding the target light and heavy chains, using linkers or adapters to provide any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0193] In one aspect, each cistron in a recombinant vector contains a secretory signal sequence component that directs translocation of the expressed polypeptide across a membrane. Generally, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize or process the signal sequence native to the heterologous polypeptide, the signal sequence is substituted with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA, and MBP. In some embodiments of the present application, the signal sequence used in both cistrons of the expression system is the STII signal sequence or a variant thereof.

[0194] In some embodiments, production of antibodies according to the present application can occur in the cytoplasm of the host cell and therefore does not require the presence of a secretory signal sequence within each cistron. In some embodiments, polypeptide components, e.g., the V of a first antigen-binding moiety optionally fused to a second antigen-binding moiety, Ha polypeptide encoding a domain and a V of a first antigen-binding moiety optionally fused to a second antigen-binding moiety L The polypeptides encoding the domains are expressed, folded, and assembled to form functional antibodies in the cytoplasm. - The cytoplasmic conditions (strains) provide favorable cytoplasmic conditions for disulfide bond formation, thereby allowing proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0195] The present invention provides an expression system in which the ratio of expressed polypeptide components can be adjusted to maximize secretion and the yield of properly assembled antibodies of the present application. Such adjustment is achieved, at least in part, by simultaneously adjusting the translational strength of the polypeptide components. One technique for adjusting translational strength is disclosed in Simmons et al., U.S. Patent No. 5,840,523, which utilizes variants of the intracistron translation initiation region (TIR). For a given TIR, a series of amino acid or nucleic acid sequence variants can be generated with a range of translational strengths, thereby providing a convenient means for adjusting this factor to the desired expression level of a particular chain. TIR variants can be generated by conventional mutagenesis techniques, resulting in codon changes that can modify the amino acid sequence; however, silent changes to the nucleic acid sequence are preferred. TIR modifications can include, for example, altering the number or spacing of Shine-Dalgarno sequences in addition to modifying the signal sequence. One method for generating variant signal sequences is to generate a "codon bank" at the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be achieved by varying the third nucleotide position of each codon; in addition, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions, which can complicate the creation of a bank. This mutagenesis method is described in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

[0196] Preferably, a set of vectors is generated with a range of TIR strengths for each cistron therein. This limited set provides a comparison of the expression levels of each chain, as well as the yield of the desired protein product under various TIR strength combinations. TIR strengths are determined according to the Simmons The expression level of a reporter gene can be determined by quantifying the expression level of a reporter gene, as described in detail in U.S. Patent No. 5,840,523 by Ian B. et al. Based on the translational strength comparison, desired individual TIRs are selected to be combined in the expression vector construct of the present application.

[0197] b) Prokaryotic host cells Suitable prokaryotic host cells for expressing the antibodies of the present application include Gram-negative or Gram-positive organisms such as Archaebacteria and Eubacteria. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In some embodiments, Gram-negative cells are used. In some embodiments, E. coli cells are used as hosts in the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219, ATCC accession number 27,325) and its derivatives (genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc-fepE) degP41 kan ROther strains and their derivatives include E. coli 294 (ATCC 31,446), E. coli B, E. coli 33D3 (including strain 33D3 having the formula (U.S. Pat. No. 5,639,635)). 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative, not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, it is necessary to select an appropriate bacterium taking into account the replicability of the replicon in the bacterial cell. For example, when a well-known plasmid such as pBR322, pBR325, pACYC177, or pKN410 is used to provide the replicon, E. coli, Serratia, or Salmonella species can be suitably used as the host.

[0198] Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0199] c) Protein production Host cells are transformed with the above-described expression vector and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the gene encoding the desired sequence. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA is replicable either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is generally used for bacterial cells that contain substantial cell wall barriers. Another method of transformation uses polyethylene glycol / DMSO. Yet another technique that can be used is electroporation.

[0200] Prokaryotic cells used to produce the antibodies of the present application are grown in media known in the art and suitable for culturing the selected host cells. An example of a suitable medium is Luria Broth (LB) containing necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing an ampicillin resistance gene.

[0201] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may also be included at appropriate concentrations, introduced alone or in mixtures with other supplements or media, such as complex nitrogen sources. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol, and dithiothreitol.

[0202] Prokaryotic host cells are cultured at a suitable temperature. For example, for E. coli growth, preferred temperatures range from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, and even more preferably about 30°C. The pH of the medium can be any pH in the range of about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0203] When an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one embodiment of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, the transformed host cell is cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-147). Various other inducers can be used according to the vector construction used, as known in the art.

[0204] The expressed antibodies of the present application are secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves disruption of the microorganism, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further isolated and characterized using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0205] Alternatively, protein production is carried out in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation techniques are available for the production of recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermentors with a volume of approximately 100 liters or less, and can range from about 1 liter to about 100 liters.

[0206] During the fermentation process, induction of protein expression is typically initiated when cells reach a desired density, e.g., an OD of about 180-220. 550 Induction begins after growth under suitable conditions until the cells reach a quiescent state, at which point they are in early stationary phase. Various inducers can be used according to the vector construct used, as known in the art. Cells can be grown for a shorter period before induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction times can also be used. It can be used.

[0207] Various fermentation conditions can be modified to improve the production yield and quality of the antibodies of the present application. For example, to improve the proper assembly and folding of secreted polypeptides, prokaryotic host cells can be co-transformed with an additional vector overexpressing a chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl prolyl cis, trans-isomerase with chaperone activity). Chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-19605, Georgiou et al., US Patent No. 6,083,715, Georgiou et al., US Patent No. 6,027,888, Bothmann and Pluckthun (2000) J.Biol.Chem.275:17100-17105, Ramm and Pluckthun (2000) J.Biol.Chem.275:17106-17113, Arie et al. (2001) Mol.Microbiol.39:199-210.

[0208] To minimize proteolysis of expressed heterologous proteins (especially those sensitive to proteolysis), certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to introduce genetic mutation(s) in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998) (see above), Georgiou et al., U.S. Pat. No. 5,264,365, Georgiou et al., U.S. Pat. No. 5,508,192, and Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0209] E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can be used as host cells in expression systems encoding the antibodies of the present application.

[0210] d) Protein purification The antibodies produced herein can be further purified to obtain substantially homogeneous preparations for further assays and uses. Standard protein purification methods known in the art can be used. The following techniques are exemplary of suitable purification techniques: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0211] In one embodiment, protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies containing the Fc region of the present application. Protein A is a 41 kD cell wall protein from Staphylococcus aureus that binds to the Fc region of antibodies with high affinity. Lindmark et al. (1983) J. Immunol. Meth. 62:1-13. The solid phase to which protein A is immobilized is preferably a column containing a glass or silica surface, more preferably a controlled-pore glass column or a silicic acid column. In some applications, the column is coated with a reagent such as glycerol to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0212] 4. Recombinant production in eukaryotic cells For eukaryotic expression, vector components generally include, but are not limited to, a signal sequence, an origin of replication, one or more marker genes, and one or more of an enhancer element, a promoter, and a transcription termination sequence.

[0213] a) Signal Sequence Component Vectors for use with eukaryotic hosts may also have inserts encoding a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences are available, as well as viral secretory leaders, such as the herpes simplex gD signal.

[0214] The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody of the present application.

[0215] b) Origin of replication Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

[0216] c) Selective Gene Components Expression and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency, or (c) supply essential nutrients unavailable from complex media, e.g., a gene encoding D-alanine racemase for Bacilli.

[0217] One example of a selection scheme utilizes a drug to arrest growth of the host cell. Cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0218] Other examples of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up nucleic acid encoding the antibody of the present application, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably a primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, and the like.

[0219] For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, an appropriate host cell is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0220] Alternatively, host cells transformed or co-transformed with a polypeptide-encoding DNA sequence, a wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0221] d) promoter component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding the desired polypeptide sequence. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be a signal for addition of a polyA tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0222] Other promoters suitable for use with prokaryotic hosts include the phoA promoter, the -lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the antibody.

[0223] Transcription of polypeptides from vectors in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter or immunoglobulin promoters, or heat shock promoters, provided that such promoters are compatible with the host cell system.

[0224] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), regarding expression of human interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

[0225] e) enhancer element component Transcription of a DNA encoding the antibody of the present application by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, an enhancer from a eukaryotic cell virus will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) for a discussion of enhancing elements for activation of eukaryotic promoters. The enhancer can be spliced ​​into the vector at a position 5' or 3' to the polypeptide-coding sequence, but is preferably located at a site 5' from the promoter.

[0226] f) transcription termination component Expression vectors used in eukaryotic host cells (e.g., yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the polypeptide-encoding mRNA. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94 / 11026 and the expression vector disclosed therein.

[0227] g) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human liver cancer line (Hep G2).

[0228] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0229] h) Cultivation of host cells The host cells used to produce the antibodies of the present application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Reissue No. 30,985, can be used as a culture medium for host cells. Any of these media may optionally contain hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (minerals usually present at minimal concentrations in the micromolar range), and / or other growth factors (such as insulin, transferrin, or epidermal growth factor). The culture medium may be supplemented with ATP (defined as an organic compound), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, will be those conventionally used with the host cell selected for expression and will be apparent to those skilled in the art.

[0230] i) Protein purification When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris (either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant of such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0231] Protein compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human immunoglobulins containing one, two, or four heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human 3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. The antibody is CH If the antibody contains three domains, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0232] After any preliminary purification step(s), the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0233] Immunoconjugates In some embodiments, the present application provides immunoconjugates comprising any of the antibodies (such as sdAbs) described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes. Also provided.

[0234] In some embodiments, the immunoconjugate is an antibody that binds to a maytansinoid (e.g., U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0425235). B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al. al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and antibody-drug conjugates (ADCs) conjugated to one or more drugs, including, but not limited to, CC1065.

[0235] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diansin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0236] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphy studies, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as, again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0237] Conjugates of antibodies and cytotoxic agents were prepared using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), iminothiolane (IT), and cytotoxic agents. Various bifunctional protein binding agents can be used to generate immunotoxins, such as bifunctional derivatives of isoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026. The linker may also be a "cleavable linker" that facilitates the release of the cytotoxic drug within the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Pat. No. 5,208,020) may be used.

[0238] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with crosslinker reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0239] Methods and Compositions for Diagnostics and Detection In some embodiments, any of the antibodies (such as sdAbs) provided herein are useful for detecting the presence of BCMA in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissues.

[0240] In some embodiments, an anti-BCMA antibody (such as any one of the anti-BCMA sdAbs described herein) is provided for use in a diagnostic or detection method. In a further aspect, a method of detecting the presence of BCMA in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of BCMA protein in the biological sample. In certain embodiments, the BCMA is human BCMA. In certain embodiments, the method comprises contacting the biological sample with an anti-BCMA antibody described herein under conditions that allow binding of the anti-BCMA antibody to BCMA, and detecting whether a complex is formed between the anti-BCMA antibody and BCMA. Such a method may be an in vitro method or an in vivo method. In some embodiments, the anti-BCMA antibody is used to select subjects eligible for treatment with the anti-BCMA antibody, e.g., BCMA is a biomarker for patient selection.

[0241] In certain embodiments, labeled anti-BCMA sdAbs are provided. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels) and moieties that are indirectly detected, for example, by enzymatic reaction or molecular interaction, such as enzymes or ligands. Exemplary labels include radioisotopes 32 P, 14 C. 125 I, 3 H, and 131Examples of suitable fluorophores include, but are not limited to, I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled with enzymes that oxidize dye precursors using hydrogen peroxide (such as HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0242] III. Chimeric Antigen Receptors One aspect of the present application is directed to one or more single domain antibodies (V H

[0013] Chimeric antigen receptors (CARs) are provided that comprise an extracellular antigen-binding domain comprising an anti-BCMA sdAb (e.g., H). Any one of the anti-BCMA sdAbs described in Section II may be used in the CARs described herein. Exemplary structures of CARs are shown in Figures 15A-15D.

[0243] In some embodiments, a BCMA-targeting CAR (referred to herein as a "BCMA CAR") is provided, comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or humanized. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the BCMA CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first costimulatory signaling domain derived from CD28, a second costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the BCMA CAR is monospecific. In some embodiments, the BCMA CAR is monovalent.

[0244] In some embodiments, a BCMA CAR is provided comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising an anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2; R1, CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and CDR3 comprising the amino acid sequence of SEQ ID NO: 78; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 3, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 79; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and CDR3 comprising the amino acid sequence of SEQ ID NO: 80; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 5, CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR3 comprising the amino acid sequence of SEQ ID NO: 81; ( (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 82; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and CDR3 comprising the amino acid sequence of SEQ ID NO: 83; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR3 comprising the amino acid sequence of SEQ ID NO: 84; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR3 comprising the amino acid sequence of SEQ ID NO: (10) CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 48, and CDR3 having the amino acid sequence of SEQ ID NO: 86, (11) CDR1 having the amino acid sequence of SEQ ID NO: 11, CDR2 having the amino acid sequence of SEQ ID NO: 49, and CDR3 having the amino acid sequence of SEQ ID NO: 87, (12) CDR1 having the amino acid sequence of SEQ ID NO: 12, CDR2 having the amino acid sequence of SEQ ID NO: 50, and CDR3 having the amino acid sequence of SEQ ID NO: 88, (13) CDR1 having the amino acid sequence of SEQ ID NO: 13, (14) CDR1 having the amino acid sequence of SEQ ID NO: 14, CDR2 having the amino acid sequence of SEQ ID NO: 52, and CDR3 having the amino acid sequence of SEQ ID NO: 90, (15) CDR1 having the amino acid sequence of SEQ ID NO: 15, CDR2 having the amino acid sequence of SEQ ID NO: 53, and CDR3 having the amino acid sequence of SEQ ID NO: 91, (16) CDR1 having the amino acid sequence of SEQ ID NO: 16, CDR2 having the amino acid sequence of SEQ ID NO: 54, and CDR3 having the amino acid sequence of SEQ ID NO: 92,(17) CDR1 comprising the amino acid sequence of SEQ ID NO: 17, CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR3 comprising the amino acid sequence of SEQ ID NO: 93; (18) CDR1 comprising the amino acid sequence of SEQ ID NO: 18, CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR3 comprising the amino acid sequence of SEQ ID NO: 94; (19) CDR1 comprising the amino acid sequence of SEQ ID NO: 19, CDR2 comprising the amino acid sequence of SEQ ID NO: 57, and CDR3 comprising the amino acid sequence of SEQ ID NO: 95; (20) CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 58 (21) CDR1 comprising the amino acid sequence of SEQ ID NO: 21, CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and CDR3 comprising the amino acid sequence of SEQ ID NO: 97, (22) CDR1 comprising the amino acid sequence of SEQ ID NO: 22, CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR3 comprising the amino acid sequence of SEQ ID NO: 98, (23) CDR1 comprising the amino acid sequence of SEQ ID NO: 23, CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and CDR3 comprising the amino acid sequence of SEQ ID NO: 99, (24) CDR1 comprising the amino acid sequence of SEQ ID NO: 24 (25) CDR1 comprising the amino acid sequence of SEQ ID NO: 25, CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and CDR3 comprising the amino acid sequence of SEQ ID NO: 101; (26) CDR1 comprising the amino acid sequence of SEQ ID NO: 26, CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR3 comprising the amino acid sequence of SEQ ID NO: 102; (27) CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR3 comprising the amino acid sequence of SEQ ID NO: 103 (28) CDR1 having the amino acid sequence of SEQ ID NO: 28, CDR2 having the amino acid sequence of SEQ ID NO: 66, and CDR3 having the amino acid sequence of SEQ ID NO: 104, (29) CDR1 having the amino acid sequence of SEQ ID NO: 29, CDR2 having the amino acid sequence of SEQ ID NO: 67, and CDR3 having the amino acid sequence of SEQ ID NO: 105, (30) CDR1 having the amino acid sequence of SEQ ID NO: 30, CDR2 having the amino acid sequence of SEQ ID NO: 68, and CDR3 having the amino acid sequence of SEQ ID NO: 106, (31) CDR1 having the amino acid sequence of SEQ ID NO: 31,Sequence number, (31) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO: 35 (36) a CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 112, (37) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 113, or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO: 38, a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or humanized. In some embodiments, the anti-BCMA sdAb is a V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. HIn some embodiments, the intracellular signaling domain comprises an H domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the BCMA CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first costimulatory signaling domain derived from CD28, a second costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the BCMA CAR is monospecific. In some embodiments, the BCMA CAR is monovalent.

[0245] In some embodiments, a BCMA CAR is provided comprising a polypeptide having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-256 and 298-335. In some embodiments, a BCMA CAR is provided comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-256 and 298-335. Polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-256 and 298-335 are also provided.

[0246] In some embodiments, an isolated nucleic acid encoding any of the BCMA CARs provided herein is provided. In some embodiments, the nucleic acid sequence is at least about 85%, 86%, or 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 257-297 and 336-373. 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid of SEQ ID NO: 257-297. In some embodiments, an isolated nucleic acid is provided comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 257-297 and 336-373. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA. In some embodiments, a vector is provided comprising any one of the nucleic acids encoding a BCMA CAR described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector. Exemplary monovalent BCMA CARs are shown in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3]

[0247] Multivalent Chimeric Antigen Receptor The present application also provides multivalent CARs having two or more (such as about any one of 2, 3, 4, 5, 6, or more) binding moieties that specifically bind to an antigen, such as BCMA. In some embodiments, one or more of the binding moieties are antigen-binding fragments. In some embodiments, one or more of the binding moieties comprise a single domain antibody. In some embodiments, one or more of the binding moieties are derived from a camelid antibody. In some embodiments, one or more of the binding moieties are derived from a four-chain antibody. In some embodiments, one or more of the binding moieties are scFvs. In some embodiments, one or more of the binding moieties are derived from a human antibody. In some embodiments, one or more of the binding moieties are polypeptide ligands or other non-antibody polypeptides that specifically bind to an antigen. In some embodiments, the multivalent CAR is monospecific, i.e., the multivalent CAR targets a single antigen and comprises two or more binding sites for a single antigen. In some embodiments, the multivalent CAR is multispecific, i.e., the multivalent CAR targets two or more antigens and comprises more than two binding sites for at least one antigen. Binding moieties specific for the same antigen may bind to the same epitope of the antigen (i.e., a "single epitope CAR") or may bind to different epitopes of the antigen (i.e., a "multi-epitope CAR", such as a two-epitope CAR or a three-epitope CAR). Binding moieties specific for the same antigen may comprise the same or different sdAbs.

[0248] In some embodiments, the present application provides a method for detecting a tumor antigen comprising: (a) an extracellular antigen-binding domain comprising a plurality (at least about one of 2, 3, 4, 5, 6, or more) binding moieties that specifically bind to an antigen (such as a tumor antigen); (b) a transmembrane domain; and (c) an intracellular signaling domain. and a polypeptide comprising: a chimeric antigen receptor (CA) comprising: a nucleotide sequence selected from the group consisting of ...

[0249] In some embodiments, the present application provides a multivalent (bivalent, trivalent, or higher valency) chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a plurality (at least about one of 2, 3, 4, 5, 6, or more) single domain antibodies (sdAbs) that specifically bind to an antigen (such as a tumor antigen); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77.

[0250] In some embodiments, the present application provides a multivalent (bivalent, trivalent, or higher) chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first binding moiety that specifically binds to a first epitope of an antigen (such as a tumor antigen) and a second binding moiety that specifically binds to a second epitope of the antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the first binding moiety is an sdAb and the second binding moiety is derived from a human antibody (e.g., an scFv). In some embodiments, the first binding moiety is an sdAb and the second binding moiety is a polypeptide ligand. In some embodiments, the first epitope is the same as the second epitope. In some embodiments, the first epitope is different from the second epitope. In some embodiments, the multivalent CAR specifically binds to two different epitopes on the antigen. In some embodiments, the multivalent CAR specifically binds to three or more different epitopes on the antigen.

[0251] In some embodiments, the present application provides a multivalent (bivalent, trivalent, or higher valency) chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first sdAb that specifically binds to a first epitope of an antigen (such as a tumor antigen) and a second sdAb that specifically binds to a second epitope of the antigen, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77.

[0252] In some embodiments, the binding moieties, such as sdAbs (including multiple sdAbs, or a first sdAb and / or a second sdAb) are camelid, chimeric, human, or humanized. In some embodiments, the binding moieties or sdAbs are fused to each other via a peptide bond or peptide linker. In some embodiments, each peptide linker is about 50 or less amino acids in length (such as about any one of 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain is an immune effector In some embodiments, the multivalent CAR further comprises a primary intracellular signaling domain of a human T cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the multivalent CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multivalent CAR further comprises a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N- to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the multivalent CAR is monospecific. In some embodiments, the multivalent CAR is multispecific, such as bispecific.

[0253] The multivalent CARs described herein may be particularly suitable for targeting multimeric antigens through synergistic binding by different antigen binding sites, or for enhancing binding affinity or avidity to antigens. Any of the anti-BCMA sdAbs described herein may be used in the extracellular antigen-binding domain of the multivalent CARs described herein. A list of exemplary multivalent BCMA CARs, exemplary sequences, constructs, and their vectors is provided in Table 5.

[0254] In some embodiments, multivalent BCMA-targeting CARs are provided that include (a) an extracellular antigen-binding domain that includes multiple (at least about one of two, three, four, or more) BCMA-binding moieties (e.g., anti-BCMA sdAbs), (b) a transmembrane domain, and (c) an intracellular signaling domain. Any of the anti-BCMA sdAbs can be used to construct the multivalent BCMA CAR. In some embodiments, the extracellular antigen-binding domain specifically binds to a single epitope on BCMA, and these CARs are referred to herein as single-epitope multivalent BCMA CARs.

[0255] In some embodiments, a multivalent BCMA CAR is provided comprising: (a) an extracellular antigen binding domain comprising a plurality (at least one of about 2, 3, 4, or more) anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAbs comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 77.

[0256] In some embodiments, a multivalent BCMA CAR (also referred to herein as a "multi-epitope multivalent CAR") is provided that comprises: (a) an extracellular antigen-binding domain comprising at least two (e.g., any one of 2, 3, 4, or more) BCMA-binding moieties; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the at least two BCMA-binding moieties specifically bind to at least two different epitopes on BCMA. In some embodiments, the extracellular antigen-binding domain comprises a first BCMA-binding moiety and a second BCMA-binding moiety. In some embodiments, the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is derived from a human antibody (e.g., an scFv). In some embodiments, the first BCMA-binding moiety is an sdAb and the second BCMA-binding moiety is a BCMA polypeptide ligand. In some embodiments, the first anti-BCMA-binding moiety and / or the second BCMA-binding moiety specifically bind to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs: 388-394. In some embodiments, the first BCMA binding moiety specifically binds to an epitope derived from SEQ ID NO: 389 and / or 390. In some embodiments, the second The BCMA binding portion specifically binds to an epitope derived from SEQ ID NO: 391 and / or 392.

[0257] In some embodiments, a multivalent BCMA CAR is provided comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb and the second anti-BCMA sdAb specifically bind to different epitopes on BCMA. Any of the anti-BCMA sdAbs can be used to construct the multivalent BCMA CAR. In some embodiments, the first anti-BCMA sdAb and / or the second anti-BCMA sdAb specifically bind to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs: 388-394. In some embodiments, the first anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NOs: 389 and / or 390. In some embodiments, the second anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NOs: 391 and / or 392.

[0258] In some embodiments there is provided a multivalent BCMA CAR comprising (a) an extracellular antigen binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signalling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 79, and the second anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 117. H In some embodiments, the second anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 124. H In some embodiments, the first anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 124. H In some embodiments, the second anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 117. HContains the H domain.

[0259] In some embodiments there is provided a multivalent BCMA CAR comprising (a) an extracellular antigen binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signalling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 86, and the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments the first anti-BCMA sdAb comprises a V H In some embodiments, the second anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 125. H Contains the H domain.

[0260] In some embodiments there is provided a multivalent BCMA CAR comprising (a) an extracellular antigen binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signalling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 83, and the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments the first anti-BCMA sdAb comprises a V H In some embodiments, the second anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 125. H Contains the H domain.

[0261] In some embodiments there is provided a multivalent BCMA CAR comprising (a) an extracellular antigen binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signalling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 91, and the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments the first anti-BCMA sdAb comprises a V H In some embodiments, the second anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 132. H Contains the H domain.

[0262] In some embodiments there is provided a multivalent BCMA CAR comprising (a) an extracellular antigen binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signalling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 94, and the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments the first anti-BCMA sdAb comprises a V H In some embodiments, the second anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 134. H Contains the H domain.

[0263] In some embodiments there is provided a multivalent BCMA CAR comprising (a) an extracellular antigen binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signalling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 96, and the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 comprising the amino acid sequence of SEQ ID NO: 66, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 134. H In some embodiments, the second anti-BCMA sdAb comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 142. H Contains the H domain.

[0264] In some embodiments, the first BCMA binding moiety (e.g., a first anti-BCMA sdAb) is located N-terminal to the second BCMA binding moiety (e.g., a second anti-BCMA sdAb). In some embodiments, the first BCMA binding moiety (e.g., a first anti-BCMA sdAb) is located C-terminal to the second BCMA binding moiety (e.g., a second anti-BCMA sdAb). In some embodiments, the first BCMA binding moiety (e.g., a first anti-BCMA sdAb) and the second BCMA binding moiety (e.g., a second anti-BCMA sdAb) are fused to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker is about 50 or less amino acids in length (such as about any one of 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the multivalent BCMA CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multivalent BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the multivalent BCMA CAR is bivalent. In some embodiments, the multivalent BCMA CAR is trivalent. In some embodiments, the multivalent BCMA CAR specifically binds to two different epitopes on BCMA. In some embodiments, the multivalent BCMA CAR specifically binds to three or more different epitopes on BCMA.

[0265] In some embodiments, a multivalent BCMA CAR is provided comprising a polypeptide having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 298-335. In some embodiments, a multivalent BCMA CAR is provided comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298-335. Polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298-335 are also provided.

[0266] In some embodiments, an isolated nucleic acid encoding any of the multivalent BCMA CARs provided herein is provided. In some embodiments, an isolated nucleic acid is provided that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 336-373. In some embodiments, an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 336-373 is provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA. In some embodiments, a vector is provided that comprises any one of the nucleic acids encoding the multivalent BCMA CARs described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector. Exemplary multivalent BCMA CARs are shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0267] Multispecific chimeric antigen receptors The present application further provides multispecific chimeric antigen receptors that target two or more (such as about any one of 2, 3, 4, 5, 6, or more) different antigens. In some embodiments, the multispecific CAR has one antigen-binding site for each antigen. In some embodiments, the multispecific CAR has more than two binding sites for at least one antigen. Each antigen-binding site may comprise an sdAb. For example, in some embodiments, the multispecific CAR is a bispecific CAR that comprises an extracellular antigen-binding domain comprising two different sdAbs that each specifically bind to an antigen. In some embodiments, the multispecific CAR is a trispecific CAR that comprises an extracellular antigen-binding domain comprising three different sdAbs that each specifically bind to an antigen.

[0268] In some embodiments, a multispecific (bispecific) chimeric antigen receptor (CAR) is provided, comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first single domain antibody (sdAb) that specifically binds BCMA and a second single domain antibody (sdAb) that specifically binds a second antigen (such as a tumor antigen), (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the first antigen is different from the second antigen. In some embodiments, the second antigen is one of CD19, CD20, CD22, CD33, CD38, CS1, or a combination thereof. , ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the first sdAb and / or second sdAb are camelid, chimeric, human, or humanized. In some embodiments, the first sdAb and second sdAb are fused to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker is about 50 or less amino acids in length (such as about any one of 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the multispecific CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multispecific CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ.In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a costimulatory signaling domain derived from CD28, and a primary intracellular signaling domain derived from CD3ζ.

[0269] Extracellular antigen-binding domain The extracellular antigen-binding domain of a CAR described herein comprises one or more (such as any one of 1, 2, 3, 4, 5, 6, or more) binding moieties, such as sdAbs. In some embodiments, one or more binding moieties are antibodies or antigen-binding fragments thereof. In some embodiments, one or more binding moieties are derived from a four-chain antibody. In some embodiments, one or more binding moieties are derived from a camelid antibody. In some embodiments, one or more binding moieties are derived from a human antibody. In some embodiments, one or more binding moieties are non-antibody binding proteins, e.g., polypeptide ligands, or engineered proteins that bind to antigens. Binding moieties can be directly fused to each other via peptide bonds or peptide linkers.

[0270] 1. Single Domain Antibodies In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising one or more sdAbs. The sdAbs may be of the same or different origin and may be of the same or different size. Exemplary sdAbs include heavy chain-only antibodies (e.g., V H H or V NAR ), a binding molecule naturally devoid of light chains, a single domain (V) derived from a conventional four-chain antibody H or V LThese include, but are not limited to, humanized heavy chain-only antibodies, human sdAbs produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. Any sdAb known in the art or developed by the inventors can be used to construct the CARs described herein, including the sdAbs described in Section II of this application. sdAbs can be derived from mouse, rat, human, camel, They may be derived from any species, including, but not limited to, llama, lamprey, fish, shark, goat, rabbit, and cow. Single domain antibodies contemplated herein also include naturally occurring sdAbs from species other than camelids and sharks.

[0271] In some embodiments, the variable domains are derived from naturally occurring single domain antigen binding molecules known as heavy chain antibodies that are devoid of light chains (also referred to herein as "heavy chain-only antibodies"). Such single domain molecules are disclosed, for example, in WO 94 / 04678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, variable domains derived from heavy chain molecules that naturally lack light chains are referred to as the conventional V of four-chain immunoglobulins. H To distinguish it from V H H. H H molecules can be derived from antibodies produced in Camelidae species, such as camel, llama, vicuna, dromedary, alpaca, and guanaco. Other non-Camelidae species can produce heavy chain molecules that naturally lack light chains, and such V H H is within the scope of this application.

[0272] V from the Camelidae family H H molecules are about 10 times smaller than IgG molecules. They are single polypeptides, very stable, and can withstand extreme pH and temperature conditions. Furthermore, they can resist protease action, which is not the case for conventional four-chain antibodies. Furthermore, V HIn vitro expression of H produces high yields of properly folded, functional V H In addition, antibodies generated in camelids can recognize epitopes other than those recognized by antibodies generated in vitro by the use of antibody libraries or by immunization of mammals other than camelids (see, for example, WO9749805). Thus, one or more V H Multispecific or multivalent CARs containing H domains can interact with targets more efficiently than multispecific or multivalent CARs containing antigen-binding fragments derived from conventional four-chain antibodies. H Since H is known to bind to "unusual" epitopes such as cavities or grooves, H The affinity of H-containing CARs may be more suitable for therapeutic treatment than conventional multispecific polypeptides.

[0273] In some embodiments, sdAbs are derived from the variable region of immunoglobulins found in cartilaginous fish. For example, sdAbs can be derived from the immunoglobulin isotype known as the novel antigen receptor (NAR) found in shark serum. Methods for producing single domain molecules derived from the variable region of NARs ("IgNARs") are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0274] In some embodiments, sdAbs are recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or in vitro generated (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions may be modified by "camelization" of specific amino acid residues within the framework regions. Camelization involves the modification of (naturally occurring) V sequences from traditional four-chain antibodies. H V of a heavy chain antibody of one or more amino acid residues within the amino acid sequence of the domain HThis refers to the replacement or substitution of one or more of the amino acid residues occurring at the corresponding position(s) in the H domain. This can be done in a manner known per se, which will become clear to those skilled in the art based on the further description herein. Such "camelizing" substitutions are preferably made in the V H -V L Insertions are made at amino acid positions that form and / or are present at the interface and / or at the so-called Camelidae signature residues as defined herein (see, e.g., WO94 / 04678, Davies and Riechmann FEBS Letters 339:285-290,1994, Davies and Riechmann Protein Engineering 9(6):531-537,1996, Riechmann J.Mol.Biol.259:957-969,1996, and Riechmann and Muylde (See, e.g., R. Rmans J. Immunol. Meth. 231:25-38, 1999).

[0275] In some embodiments, the sdAb is a human sdAb produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794. In some embodiments, the sdAb is affinity matured.

[0276] In some embodiments, a naturally occurring V against a particular antigen or target H The H domain is a member of the Camelidae V HThe antigen or target may be obtained from a (natural or immune) library of H sequences. Such methods may or may not involve screening such libraries using the antigen or target, or at least one portion, fragment, antigenic determinant, or epitope thereof, using one or more screening techniques known per se. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, the (natural or immune) V sequences may be isolated by techniques such as random mutagenesis and / or CDR shuffling, as described, for example, in WO 00 / 43507. H V obtained from the H library H H libraries (natural or immune) H Improved synthetic or semi-synthetic libraries derived from the H library can be used.

[0277] In some embodiments, the sdAb is generated from a traditional four-chain antibody. 0 368 684, Ward et al. (Nature 1989 Oct. 12;341(6242):544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490, WO06 / 030220, and WO06 / 003388.

[0278] 2. Antigen The antigen(s) targeted by the CAR of the present application are cell surface molecules. The binding moiety (such as an sdAb) can be selected to recognize an antigen that serves as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen (such as a first antigen and / or a second antigen) is a tumor antigen. In some embodiments, a multispecific CAR targets two or more tumor antigens. In some embodiments, the tumor antigen is associated with a B-cell malignancy. Tumors express several proteins that can serve as target antigens for the immune response, specifically, the T-cell-mediated immune response. The antigen targeted by the CAR can be an antigen on a single diseased cell or an antigen expressed on different cells that each contribute to the disease. The antigen targeted by the CAR can be directly or indirectly involved in the disease.

[0279] Tumor antigens are proteins produced by tumor cells that can elicit an immune response, specifically a T cell-mediated immune response. The choice of targeted antigen of the present invention will depend on the particular type of cancer being treated. Exemplary tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0280] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate target antigens in B-cell lymphomas.

[0281] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur in other cells in the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. The expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they can be antigens that are usually present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0282] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO- 1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0283] In some embodiments, the antigen (e.g., the first antigen and / or the second antigen) is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77.

[0284] 3. Peptide Linker Various binding moieties (sdAbs, etc.) in the multispecific or multivalent CARs described herein can be fused to each other via a peptide linker. In some embodiments, binding moieties (such as sdAbs) are fused directly to each other without any peptide linker. The peptide linkers connecting different binding moieties (such as sdAbs) can be the same or different. Different domains of a CAR can also be fused to each other via a peptide linker.

[0285] Each peptide linker in a CAR can have the same or different length and / or sequence depending on the structural and / or functional characteristics of the sdAb and / or various domains. Each peptide linker can be independently selected and optimized. The length, flexibility, and / or other properties of the peptide linker(s) used in a CAR can have some effect on properties, including, but not limited to, affinity, specificity, or avidity for one or more specific antigens or epitopes. For example, a longer peptide linker can be selected to ensure that two adjacent domains do not sterically interfere with each other. For example, in a multivalent or multispecific CAR of the present application that includes sdAbs directed against a multimeric antigen, the length and flexibility of the peptide linker are preferably such that each sdAb in the multivalent CAR can bind to an antigenic determinant on each subunit of the multimer. In some embodiments, a short peptide linker can be placed between the transmembrane domain and the intracellular signaling domain of the CAR. In some embodiments, the peptide linker contains flexible residues (such as glycine and serine) to allow adjacent domains to move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker.

[0286] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids in length. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less amino acids in length. In some embodiments, the length of the peptide linker is any of about 1 to about 10 amino acids, about 1 to about 20 amino acids, about 1 to about 30 amino acids, about 5 to about 15 amino acids, about 10 to about 25 amino acids, about 5 to about 30 amino acids, about 10 to about 30 amino acids in length, about 30 to about 50 amino acids, about 50 to about 100 amino acids, or about 1 to about 100 amino acids.

[0287] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n , (GGGS) n , and (GGGGS) nwhere n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence GGGGS (SEQ ID NO:208), (GGGGS)2 (SEQ ID NO:209), (GGGS)4 (SEQ ID NO:210), GGGGSGGGGSGGGGGGSGSGGGGS (SEQ ID NO:211), GGGGSGGGGSGGGGGGSGSGGGGSGGGGSGGGGS (SEQ ID NO:212), (GGGGS)3 (SEQ ID NO:213), (GGGGS)4 (SEQ ID NO:214), or (GGGGS)3 (SEQ ID NO:215).

[0288] Transmembrane domain The CAR of the present application is a transmembrane CAR that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain comprises a domain. The transmembrane domain can be derived from either natural or synthetic sources.As used herein, " transmembrane domain " refers to any protein structure that is thermodynamically stable in a cell membrane, preferably in a eukaryotic cell membrane.The transmembrane domain that is suitable for use in the CAR described herein can be obtained from naturally occurring proteins.Alternatively, it can be a synthetic non-naturally occurring protein segment, for example, a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0289] Transmembrane domains are classified based on their three-dimensional structure. For example, transmembrane domains can form an alpha helix, a complex of two or more alpha helices, a beta barrel, or any other stable structure that can span the phospholipid bilayer of a cell. Furthermore, transmembrane domains can additionally or alternatively be classified based on their topology, including the number of times the transmembrane domain crosses the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, while multi-pass membrane proteins cross the cell membrane at least twice (e.g., two, three, four, five, six, seven, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their terminals relative to the inside and outside of the cell and the topology of the membrane-spanning segment(s). Type I membrane proteins have a single membrane-spanning region and are oriented so that the N-terminus of the protein is present on the extracellular side of the cell's lipid bilayer and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single membrane-spanning region, but are oriented so that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple membrane-spanning segments and can be further subclassified based on the number of transmembrane segments and the location of the N- and C-termini.

[0290] In some embodiments, the transmembrane domain of the CARs described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may also be compatible for use in the CARs described herein. Multi-pass membrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7, or more) alpha-helical or beta-sheet structures. Preferably, the N- and C-termini of the multi-pass membrane protein are on opposite sides of the lipid bilayer, e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is on the extracellular side.

[0291] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT In some embodiments, the transmembrane domain comprises a transmembrane domain selected from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, LyI08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from CD8α, CD4, CD28, CD137, CD80, CD86, CD 152, and PD1.

[0292] In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain is the transmembrane domain of CD28 comprising the amino acid sequence of SEQ ID NO: 194. In some embodiments, the transmembrane domain of CD28 is encoded by the nucleic acid sequence of SEQ ID NO: 203.

[0293] In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is a CD8α transmembrane domain comprising the amino acid sequence of SEQ ID NO: 193. In some embodiments, the CD8α transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 202.

[0294] The transmembrane domain for use in the CAR described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example, U.S. Patent No. 7,052,906 B1 and PCT Publication No. WO2000 / 032776 A2, the relevant disclosures of which are incorporated herein by reference.

[0295] The transmembrane domain may comprise a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids, and in some embodiments, aids in orienting the transmembrane domain within the lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0296] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine can be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises primarily hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, such as Kyte and Doolittle hydropathy analysis.

[0297] Intracellular signaling domains The CAR of the present application comprises an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one of the normal effector functions of immune effector cells expressing the CAR. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "cytoplasmic signaling domain" refers to a portion of a protein that transduces an effector function signal and instructs the cell to perform a specialized function. Typically, the entire cytoplasmic signaling domain can be used, but many In such cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such truncated portion can be used in place of the intact chain, so long as it transduces an effector function signal. Thus, the term cytoplasmic signaling domain is intended to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce an effector function signal.

[0298] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. As used herein, "ITAM" refers to a conserved protein motif typically present in the tails of signaling molecules expressed in many immune cells. This motif may comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid resulting in the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs within signaling molecules are required for intracellular signal transduction, which is mediated, at least in part, by phosphorylation of tyrosine residues within the ITAM following activation of the signaling molecule. ITAMs may also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3ζ, FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0299] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3ζ. In some embodiments, the primary intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3ζ. In some embodiments, the primary intracellular signaling domain of wild-type CD3ζ comprises the amino acid sequence of SEQ ID NO: 197. In some embodiments, the primary intracellular signaling domain is a functional variant of the cytoplasmic signaling domain of CD3ζ comprising one or more mutations, such as Q65K. In some embodiments, the primary intracellular signaling domain of mutant CD3ζ comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the primary intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 206 or 207.

[0300] Costimulatory signaling domain Many immune effector cells require costimulation in addition to antigen-specific signal stimulation to promote cell proliferation, differentiation, and survival and activate cellular effector functions. In some embodiments, a CAR comprises at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response, such as effector function. The costimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a costimulatory protein that transduces signals and regulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival.

[0301] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about any of two, three, four, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domain, for example, two copies of the costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ) are fused to each other via any peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0302] Activation of a costimulatory signaling domain within a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be compatible for use in the CARs described herein. The type(s) of costimulatory signaling domain are selected based on factors such as the type of immune effector cell (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil) on which the effector molecule is expressed and the desired immune effector function (e.g., ADCC effect). Examples of costimulatory signaling domains for use in CARs include members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6), members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF), and the like. R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-alpha / TNF-beta, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-alpha, and TNFRII / TNFRSF1B), members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150), and any other costimulatory molecules, e.g., CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha 4 / CD49d, integrin alpha 4 beta 1, integrin alpha 4 beta 7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (L The cytoplasmic signaling domain of a costimulatory protein may be, but is not limited to, FA-1, and NKG2C.

[0303] In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of a ligand that specifically binds to CD27, CD28, 4-1BB, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0304] In some embodiments, the intracellular signaling domain in a CAR of the present application comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain comprises the costimulatory signaling domain of CD28 comprising the amino acid sequence of SEQ ID NO: 195. In some embodiments, the costimulatory signaling domain of CD28 is encoded by the nucleic acid sequence of SEQ ID NO: 204.

[0305] In some embodiments, the intracellular signaling domain in a CAR of the present application comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises the costimulatory signaling domain of CD137 comprising the amino acid sequence of SEQ ID NO: 196. In some embodiments, the costimulatory signaling domain of CD137 is encoded by the nucleic acid sequence of SEQ ID NO: 205.

[0306] In some embodiments, the intracellular signaling domain in a CAR of the present application comprises the costimulatory signaling domain of CD28 and the costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ, the costimulatory signaling domain of CD28, and the costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and the cytoplasmic signaling domain of CD3ζ. In some embodiments, the costimulatory signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO: 195. In some embodiments, the costimulatory signaling domain of CD137 comprises the amino acid sequence of SEQ ID NO: 196.

[0307] Mutations of any of the costimulatory signaling domains described herein are also within the scope of the present disclosure, as they can regulate the immune response of immune cells. In some embodiments, the costimulatory signaling domain contains up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to its wild-type counterpart. Such costimulatory signaling domains containing one or more amino acid variations may be referred to as mutant forms. Mutations of amino acid residues in the costimulatory signaling domain may result in increased signal transduction and enhanced stimulation of the immune response compared to a costimulatory signaling domain that does not contain the mutation. Mutations of amino acid residues in the costimulatory signaling domain may result in decreased signal transduction and reduced stimulation of the immune response compared to a costimulatory signaling domain that does not contain the mutation.

[0308] Hinge Area The CAR of the present application may comprise a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is an amino acid segment typically found between two domains of a protein, which allows for the flexibility of the protein and the ability of one or both of those domains to function. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0309] The hinge domain can contain about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain can be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0310] In some embodiments, the hinge domain is the hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art to contain a hinge domain is compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of the hinge domain of a naturally occurring protein, conferring flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment of the hinge domain of CD8α containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids. In some embodiments, the hinge domain of CD8α comprises the amino acid sequence of SEQ ID NO: 192. In some embodiments, the hinge domain of CD8α is encoded by the nucleic acid sequence of SEQ ID NO: 201.

[0311] Hinge domains of antibodies, such as IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, the hinge domain is a hinge domain connecting antibody constant domains CH1 and CH2. In some embodiments, the hinge domain is of an antibody comprising an antibody hinge domain and one or more antibody constant regions. In some embodiments, the hinge domain comprises an antibody hinge domain and an antibody CH3 constant region. In some embodiments, the hinge domain comprises an antibody hinge domain and antibody CH2 and CH3 constant regions. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0312] Non-naturally occurring peptides can also be used as hinge domains in the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker, e.g., a (GxS)n linker, where x and n can independently be integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0313] signal peptide The CARs of the present application may include a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of the cell, allowing for incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides comprising signal sequences of naturally occurring proteins or synthetic, non-naturally occurring signal sequences compatible for use in the CARs described herein are well known in the art. It will be apparent to those skilled in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the signal peptide of CD8α comprises the amino acid sequence of SEQ ID NO: 191. In some embodiments, the signal peptide of CD8α is encoded by the nucleic acid sequence of SEQ ID NO: 199 or 200.

[0314] IV. Engineered Immune Effector Cells Further provided in the present application are host cells (such as immune effector cells) comprising any one of the CARs described herein.

[0315] Thus, in some embodiments, an engineered immune effector cell (such as a T cell) is provided that comprises a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA binding moiety that specifically binds to a first epitope of BCMA and a second BCMA binding moiety that specifically binds to a second epitope of BCMA; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different.

[0316] In some embodiments, engineered immune effector cells (such as T cells) are provided that comprise a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb that specifically binds to a first epitope of BCMA and a second anti-BCMA sdAb that specifically binds to a second epitope of BCMA; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different. In some embodiments, the first anti-BCMA sdAb and / or the second anti-BCMA sdAb are camelid, chimeric, human, or humanized. In some embodiments, the first anti-BCMA and second anti-BCMA are fused to each other via a peptide bond or peptide linker. In some embodiments, the peptide linker is about 50 or less amino acids in length (such as about any one of 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the multivalent CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multivalent CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide.In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the engineered immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the engineered immune effector cells are autologous. In some embodiments, the engineered immune effector cells are allogeneic.

[0317] In some embodiments, (a) an extracellular antigen binding domain comprising an anti-BCMA sdAb and (b) a transmembrane domain; and (c) an intracellular signaling domain. The anti-BCMA sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 79; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4. (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 5, CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR3 comprising the amino acid sequence of SEQ ID NO: 81; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 82; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 45; and CDR3 comprising the amino acid sequence of SEQ ID NO: 83; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR3 comprising the amino acid sequence of SEQ ID NO: 84; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR3 comprising the amino acid sequence of SEQ ID NO: 85; (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 10, CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and CDR3 comprising the amino acid sequence of SEQ ID NO: 86; (11) ) CDR1 comprising the amino acid sequence of SEQ ID NO: 11, CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and CDR3 comprising the amino acid sequence of SEQ ID NO: 87; (12) CDR1 comprising the amino acid sequence of SEQ ID NO: 12, CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and CDR3 comprising the amino acid sequence of SEQ ID NO: 88; (13) CDR1 comprising the amino acid sequence of SEQ ID NO: 13, CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (14) CDR1 comprising the amino acid sequence of SEQ ID NO: 14;CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (15) CDR1 comprising the amino acid sequence of SEQ ID NO: 15, CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and CDR3 comprising the amino acid sequence of SEQ ID NO: 91; (16) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and CDR3 comprising the amino acid sequence of SEQ ID NO: 92; (17) CDR1 comprising the amino acid sequence of SEQ ID NO: 17, CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR3 comprising the amino acid sequence of SEQ ID NO: 93 (18) CDR1 comprising the amino acid sequence of SEQ ID NO: 18, CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR3 comprising the amino acid sequence of SEQ ID NO: 94; (19) CDR1 comprising the amino acid sequence of SEQ ID NO: 19, CDR2 comprising the amino acid sequence of SEQ ID NO: 57, and CDR3 comprising the amino acid sequence of SEQ ID NO: 95; (20) CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and CDR3 comprising the amino acid sequence of SEQ ID NO: 96; (21) CDR1 comprising the amino acid sequence of SEQ ID NO: 21, CDR2 comprising the amino acid sequence of SEQ ID NO: 59 (22) CDR1 having the amino acid sequence of SEQ ID NO: 22, CDR2 having the amino acid sequence of SEQ ID NO: 60, and CDR3 having the amino acid sequence of SEQ ID NO: 98; (23) CDR1 having the amino acid sequence of SEQ ID NO: 23, CDR2 having the amino acid sequence of SEQ ID NO: 61, and CDR3 having the amino acid sequence of SEQ ID NO: 99; (24) CDR1 having the amino acid sequence of SEQ ID NO: 24, CDR2 having the amino acid sequence of SEQ ID NO: 62, and CDR3 having the amino acid sequence of SEQ ID NO: 100; (25) CDR1 having the amino acid sequence of SEQ ID NO: 25 (26) CDR1 comprising the amino acid sequence of SEQ ID NO: 26, CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR3 comprising the amino acid sequence of SEQ ID NO: 102; (27) CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR3 comprising the amino acid sequence of SEQ ID NO: 103; (28) CDR1 comprising the amino acid sequence of SEQ ID NO: 28, CDR2 comprising the amino acid sequence of SEQ ID NO: 66, and SEQ ID NO: 10; (29) CDR1 comprising the amino acid sequence of SEQ ID NO: 29, CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and CDR3 comprising the amino acid sequence of SEQ ID NO: 105, (30) CDR1 comprising the amino acid sequence of SEQ ID NO: 30, CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and CDR3 comprising the amino acid sequence of SEQ ID NO: 106, (31) CDR1 comprising the amino acid sequence of SEQ ID NO: 31, CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and CDR3 comprising the amino acid sequence of SEQ ID NO: 107, (32) CDR1 comprising the amino acid sequence of SEQ ID NO: 32, CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and CDR3 comprising the amino acid sequence of SEQ ID NO: 108, (33) CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and CDR3 comprising the amino acid sequence of SEQ ID NO: 109, (34) CDR1 comprising the amino acid sequence of SEQ ID NO: 34, CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and CDR3 comprising the amino acid sequence of SEQ ID NO: 110, (35) CDR1 comprising the amino acid sequence of SEQ ID NO: 35, CDR2 comprising the amino acid sequence of SEQ ID NO: 73, and CDR3 comprising the amino acid sequence of SEQ ID NO: 111, (36) CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and CDR3 comprising the amino acid sequence of SEQ ID NO: 112, (37) CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR3 comprising the amino acid sequence of SEQ ID NO: 113, or (38) CDR1 comprising the amino acid sequence of SEQ ID NO: 38, CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and CDR3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the extracellular antigen-binding domain comprises at least two anti-BCMA sdAbs. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or humanized. In some embodiments, the anti-BCMA sdAb is a V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. HIn some embodiments, the intracellular signaling domain comprises an H domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the BCMA CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the ...

Claims

1. A chimeric antigen receptor (CAR) comprising a polypeptide, (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety and a second BCMA-binding moiety, wherein the first BCMA-binding moiety is a first anti-BCMA single domain antibody (sdAb), the second BCMA-binding moiety is a second anti-BCMA sdAb, and the first anti-BCMA sdAb and the second anti-BCMA sdAb are each a VHH domain; (b) a transmembrane domain; and (c) an intracellular signaling domain; the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO:

124. comprising CDR1, CDR2 and CDR3 as defined in the sdAb, the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO:

117. comprising CDR1, CDR2 and CDR3 as defined in the sdAb, The CAR, wherein the CDR1, CDR2, and CDR3 are defined by the Kabat, AbM, Chothia, or contact numbering system.

2. The CAR described in claim 1, wherein the CDR1, CDR2 and CDR3 are defined by the Kabat numbering system.

3. A CAR described in claim 1 or 2, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

4. A CAR as described in claim 3, wherein the transmembrane domain is derived from CD8α or CD28.

5. A CAR described in any one of claims 1 to 4, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

6. A CAR as described in claim 5, wherein the primary intracellular signaling domain is derived from CD3ζ.

7. A CAR described in any one of claims 1 to 6, wherein the intracellular signaling domain includes a costimulatory signaling domain.

8. The CAR of claim 7, wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof.

9. A CAR as described in claim 8, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

10. A CAR described in any one of claims 1 to 9, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

11. A CAR as described in claim 10, wherein the hinge domain is derived from CD8α.

12. A CAR described in any one of claims 1 to 11, further comprising a signal peptide located at the N-terminus of the polypeptide.

13. A CAR as described in claim 12, wherein the signal peptide is derived from CD8α.

14. An anti-BCMA single domain antibody (sdAb), comprising: (1) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 117; or (2) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 124 Including, The anti-BCMA sdAb, wherein the CDR1, CDR2 and CDR3 are defined by the Kabat, AbM, Chothia, or contact numbering system.

15. The anti-BCMA sdAb described in claim 14, wherein the anti-BCMA sdAb is a VHH domain.

16. A chimeric antigen receptor (CAR) comprising a polypeptide, (a) an extracellular antigen-binding domain comprising at least one anti-BCMA sdAb of claim 14 or 15; (b) a transmembrane domain; and (c) an intracellular signaling domain.

17. The CAR described in claim 16, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

18. A CAR as described in claim 17, wherein the transmembrane domain is derived from CD8α or CD28.

19. A CAR described in any one of claims 16 to 18, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

20. The CAR described in claim 19, wherein the primary intracellular signaling domain is derived from CD3ζ.

21. A CAR described in any one of claims 16 to 20, wherein the intracellular signaling domain includes a costimulatory signaling domain.

22. The CAR of claim 21, wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof.

23. A CAR as described in claim 22, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

24. A CAR described in any one of claims 16 to 23, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

25. A CAR as described in claim 24, wherein the hinge domain is derived from CD8α.

26. A CAR described in any one of claims 16 to 25, further comprising a signal peptide located at the N-terminus of the polypeptide.

27. ​​A CAR as described in claim 26, wherein the signal peptide is derived from CD8α.

28. An isolated nucleic acid comprising a nucleic acid sequence encoding a CAR described in any one of claims 1 to 13 and 16 to 27, and an anti-BCMA sdAb described in claim 14 or 15.

29. The isolated nucleic acid described in claim 28, wherein the isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 155, 162, 259, 266, 269, 270, and 344-349.

30. A vector comprising the isolated nucleic acid described in claim 28 or 29.

31. An engineered immune effector cell comprising a CAR described in any one of claims 1 to 13 and 16 to 27, or an isolated nucleic acid described in claim 28 or 29, or a vector described in claim 30.

32. The engineered immune effector cell of claim 31, wherein the immune effector cell is a T cell.

33. A pharmaceutical composition comprising the engineered immune effector cells of claim 31 or 32 and a pharmaceutically acceptable carrier.

34. Use of the engineered immune effector cells of claim 31 or 32 in the manufacture of a medicament for treating cancer in an individual, wherein the cancer is a cancer comprising cells expressing BCMA.

35. The use described in claim 34, wherein the cancer is multiple myeloma.

36. The use described in claim 35, wherein the cancer is refractory or recurrent multiple myeloma.

Images