Chimeric antigen receptor targeting BCMA and method of using the same

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 of International Patent Application No. PCT / CN2016 / 094408, filed on August 10, 2016, the content of which is hereby incorporated by reference in its entirety into this specification.

[0002] Submission of Sequence Listing in ASCII text file The content of the following submission in ASCII text file is hereby incorporated by reference in its entirety into this specification: Sequence Listing in computer - readable format (CRF) (file name: 761422000640SEQLISTING.txt, recording date: August 10, 2017, size: 520KB).

[0003] The present invention relates to single - domain antibodies targeting BCMA, chimeric antigen receptors, and engineered immune effector cells, and methods of using the same.

Background Art

[0004] With the development of tumor immunotherapy and clinical techniques, chimeric antigen receptor T - cell (CAR - T) immunotherapy is currently one of the most promising approaches to tumor immunotherapy. 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 include a single - chain variable fragment (scFv) that targets a specific tumor antigen. The CAR can be expressed on the surface of T cells using gene transfection techniques. When binding to the target tumor antigen, the CAR can activate T cells to initiate a specific anti - tumor response in an antigen - dependent manner without being restricted 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 functional antigen-binding fragment known, 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 present clinically 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 every 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 has been shown to be effective in treating 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. There is a need for effective immunotherapy agents for the treatment of multiple myeloma.

[0007] The LCAR-B38M disclosed in the present invention is a bispecific BCMA targeting CAR-T, for which clinical advantages have already been shown in clinical trials regarding both safety and efficacy in the treatment of patients with refractory or relapsed multiple myeloma. In the initial clinical trial, 33 out of 35 patients (94%) had a clinical remission of multiple myeloma when receiving LCAR-B38M CAR-T cells. Most patients had only mild side effects. The study was presented by the principal inventors at both the 2017 ASCO Annual Meeting (Abstract LBA3001) and a press conference that mobilized extensive media coverage (http: / / www.ascopost.com / News / 55713).

[0008] Overall, the objective response rate was 100%, and 33 patients (94%) had an obvious clinical remission of myeloma (complete response, very good partial response, or partial response) within 2 months of receiving CAR T cells. After following this group over a period exceeding 4 months, in terms of efficacy, 14 out of 19 patients reached stringent complete response criteria, 1 patient reached partial response, and 4 patients achieved very good partial remission criteria.

[0009] The excellent efficacy and safety profiles obtained from the LCAR-B38M clinical trial are significantly superior to several other BCMA CAR-T trials reported simultaneously at ASCO, which were widely recognized as "revolutionary leaps forward" in the field of immunotherapy. It should be noted that all of 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 referred to herein are hereby incorporated by reference in their entirety.

Summary of the Invention

[0011] This application relates to an anti-BCMA single-domain antibody (sdAb), one or more anti-BCMA sdAbs (V HProvided are chimeric antigen receptors (CARs) comprising, e.g., an H fragment, engineered immune effector cells, and methods of using the same in cancer immunotherapy.

[0012] One aspect of the present application provides an anti-BCMA sdAb comprising any one CDR region of SEQ ID NOs: 115 to 152. In some embodiments, the anti-BCMA sdAb is as follows: (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 2, 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: 85 and comprising CDR3, CDR1 containing the amino acid sequence of SEQ ID NO: 10, CDR2 containing the amino acid sequence of SEQ ID NO: 48, and CDR3 containing the amino acid sequence of SEQ ID NO: 86, (11) CDR1 containing the amino acid sequence of SEQ ID NO: 11, CDR2 containing the amino acid sequence of SEQ ID NO: 49, and CDR3 containing the amino acid sequence of SEQ ID NO: 87, (12) CDR1 containing the amino acid sequence of SEQ ID NO: 12, CDR2 containing the amino acid sequence of SEQ ID NO: 50, and CDR3 containing the amino acid sequence of SEQ ID NO: 88, (13) CDR1 containing the amino acid sequence of SEQ ID NO: 13, CDR2 containing the amino acid sequence of SEQ ID NO: 51, and CDR3 containing the amino acid sequence of SEQ ID NO: 89, (14) CDR1 containing the amino acid sequence of SEQ ID NO: 14, CDR2 containing the amino acid sequence of SEQ ID NO: 52, and CDR3 containing the amino acid sequence of SEQ ID NO: 90, (15) CDR1 containing the amino acid sequence of SEQ ID NO: 15, CDR2 containing the amino acid sequence of SEQ ID NO: 53, and CDR3 containing the amino acid sequence of SEQ ID NO: 91, (16) CDR1 containing the amino acid sequence of SEQ ID NO: 16, CDR2 containing the amino acid sequence of SEQ ID NO: 54, and CDR3 containing the amino acid sequence of SEQ ID NO: 92, (17) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 55, and CDR3 containing the amino acid sequence of SEQ ID NO: 93, (18) CDR1 containing the amino acid sequence of SEQ ID NO: 18, CDR2 containing the amino acid sequence of SEQ ID NO: 56, and CDR3 containing the amino acid sequence of SEQ ID NO: 94, (19) CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 57, and CDR3 containing the amino acid sequence of SEQ ID NO: 95, (20) CDR1 containing the amino acid sequence of SEQ ID NO: 20, CDR2 containing the amino acid sequence of SEQ ID NO: 58, and CDR3 containing the amino acid sequence of SEQ ID NO: 96, (21) CDR1 containing the amino acid sequence of SEQ ID NO: 21, CDR2 containing the amino acid sequence of SEQ ID NO: 59, and CDR3 containing the amino acid sequence of SEQ ID NO: 97, (22) CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 60, and CDR3 containing the amino acid sequence of SEQ ID NO: 98, (23) CDR1 containing the amino acid sequence of SEQ ID NO: 23, CDR2 containing the amino acid sequence of SEQ ID NO: 61, and CDR3 containing the amino acid sequence of SEQ ID NO: 99, (24) CDR1 containing the amino acid sequence of SEQ ID NO: 24CDR2 containing the amino acid sequence of SEQ ID NO: 62, and CDR3 containing the amino acid sequence of SEQ ID NO: 100, (25) CDR1 containing the amino acid sequence of SEQ ID NO: 25, CDR2 containing the amino acid sequence of SEQ ID NO: 63, and CDR3 containing the amino acid sequence of SEQ ID NO: 101, (26) CDR1 containing the amino acid sequence of SEQ ID NO: 26, CDR2 containing the amino acid sequence of SEQ ID NO: 64, and CDR3 containing the amino acid sequence of SEQ ID NO: 102, (27) CDR1 containing the amino acid sequence of SEQ ID NO: 27, CDR2 containing the amino acid sequence of SEQ ID NO: 65, and CDR3 containing the amino acid sequence of SEQ ID NO: 103, (28) CDR1 containing the amino acid sequence of SEQ ID NO: 28, CDR2 containing the amino acid sequence of SEQ ID NO: 66, and CDR3 containing the amino acid sequence of SEQ ID NO: 104, (29) CDR1 containing the amino acid sequence of SEQ ID NO: 29, CDR2 containing the amino acid sequence of SEQ ID NO: 67, and CDR3 containing the amino acid sequence of SEQ ID NO: 105, (30) CDR1 containing the amino acid sequence of SEQ ID NO: 30, CDR2 containing the amino acid sequence of SEQ ID NO: 68, and CDR3 containing the amino acid sequence of SEQ ID NO: 106, (31) CDR1 containing the amino acid sequence of SEQ ID NO: 31, CDR2 containing the amino acid sequence of SEQ ID NO: 69, and CDR3 containing the amino acid sequence of SEQ ID NO: 107, (32) CDR1 containing the amino acid sequence of SEQ ID NO: 32, CDR2 containing the amino acid sequence of SEQ ID NO: 70, and CDR3 containing the amino acid sequence of SEQ ID NO: 108, (33) CDR1 containing the amino acid sequence of SEQ ID NO: 33, CDR2 containing the amino acid sequence of SEQ ID NO: 71, and CDR3 containing the amino acid sequence of SEQ ID NO: 109, (34) CDR1 containing the amino acid sequence of SEQ ID NO: 34, CDR2 containing the amino acid sequence of SEQ ID NO: 72, and CDR3 containing the amino acid sequence of SEQ ID NO: 110, (35) CDR1 containing the amino acid sequence of SEQ ID NO: 35, CDR2 containing the amino acid sequence of SEQ ID NO: 73, and CDR3 containing the amino acid sequence of SEQ ID NO: 111, (36) CDR1 containing the amino acid sequence of SEQ ID NO: 36, CDR2 containing the amino acid sequence of SEQ ID NO: 74, and CDR3 containing the amino acid sequence of SEQ ID NO: 112, (37) CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 75, and CDR3 containing the amino acid sequence of SEQ ID NO: 113, or (38) CDR1 containing the amino acid sequence of SEQ ID NO: 38A CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 114, and includes any one of them. In some embodiments, the anti-BCMA sdAb comprises a VH H domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115 to 152.

[0013] In some embodiments, an antigen-binding protein comprising an anti-BCMA heavy chain only antibody (HCAB) or any one of the anti-BCMA sdAbs described above is provided. Also provided is a BCMA epitope that 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 H fragment.

[0015] One aspect of the present 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 portion and a second BCMA-binding portion, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, one or more of the first BCMA-binding portion and the second BCMA-binding portion is an anti-BCMA sdAb. In some embodiments, the first BCMA-binding portion is a first anti-BCMA sdAb and the second BCMA-binding portion is a second anti-BCMA sdAb. In some embodiments, the first BCMA-binding portion is an anti-BCMA sdAb and the second BCMA-binding portion is derived from a human antibody. In some embodiments, the first BCMA-binding portion is an anti-BCMA sdAb and the second BCMA-binding portion is a polypeptide ligand of BCMA. In some embodiments, the first BCMA-binding portion and the second BCMA-binding portion specifically bind to the same epitope on BCMA. In some embodiments, the first BCMA-binding portion and the second BCMA-binding portion specifically bind to different epitopes on BCMA. In some embodiments, the first BCMA-binding portion and / or the second BCMA-binding portion specifically binds to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs: 388 to 394. In some embodiments, the first BCMA-binding portion specifically binds to an epitope derived from SEQ ID NO: 389 and / or 390. In some embodiments, the second BCMA-binding portion specifically binds to an epitope derived from SEQ ID NO: 391 and / or 392.

[0017] One aspect of the present application provides a multivalent (e.g., 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 NO: 389 and / or 390. In some embodiments, the second anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NO: 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 directly fused 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 amino acids in length or less (any number such as about 35, 25, 20, 15, 10, or 5 amino acids 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 the 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 co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of CD83, and combinations thereof. In some embodiments, the co-stimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137. In some embodiments, the co-stimulatory 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 includes 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 comprises the amino acid sequence of SEQ ID NO: 192.

[0023] In some embodiments according to any one of the CARs (including multivalent CARs) described above, the CAR further includes 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 present application provides the 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, and 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 provided 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, NK cell, peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, pluripotent stem cell, or 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 lymphoblastic leukemia, or chronic lymphoblastic 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 provides a pharmaceutical composition comprising any one of the anti-BCMA sdAbs described above and a pharmaceutically 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. 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 products comprising any one of the anti-BCMA sdAbs, CARs (including multivalent CARs), engineered immune effector cells, isolated nucleic acids, or vectors described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0032]

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DETAILED DESCRIPTION OF THE INVENTION

[0033] The present application provides a chimeric antigen receptor (CAR) 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 sdAb). Also provided are multivalent CARs comprising at least two binding moieties (such as sdAb) that specifically bind to a single antigen. In some embodiments, the present application provides a multivalent (bivalent or trivalent, etc.) CAR 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 sdAb, the CAR, and the engineered immune effector cells expressing the CARs described in the present application are useful agents for cancer treatment.

[0034] In particular, the present application demonstrates the excellent 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 a Phase I / II clinical trial In the interim analysis, patients with relapsed or refractory multiple myeloma responded 100% to LCAR-B38M CAR-T therapy. 94% of the patients had an obvious clinical remission of myeloma within 2 months of receiving CAR-T therapy. Patients who reached stringent complete response (sCR) criteria maintained a minimal residual disease-free state more than 1 year after receiving CAR-T therapy. Furthermore, LCAR-B38M CAR-T therapy showed good tolerance among patients because most patients experienced only mild and manageable cytokine release syndrome, which is a common side effect of CAR-T cell-based therapy. The patients did not experience neurological side effects. Relatively, a pilot clinical study of monovalent CAR containing a single anti-BCMA sdAb showed lower objective response rates and complete remission rates among the treated patients, as well as higher recurrence rates. 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 CAR of this application are unexpected.

[0035] Accordingly, one aspect of this 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 that comprises 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] Novel anti-BCMA sdAbs and CARs comprising any one or more of the anti-BCMA sdAbs described herein are further provided.

[0038] Pharmaceutical compositions, kits, products, and methods for treating cancer using engineered immune effector cells (such as T cells) comprising a CAR, engineered immune effector cells, or sdAbs are also described herein.

[0039] I. Definitions 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 having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab’)2, 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 in addition to an additional polypeptide called the J chain and contain ten antigen-binding sites, while IgA antibodies are composed of two to five basic four-chain units that can polymerize to form a multivalent aggregate in combination with the J chain. In the case of IgG, the four-chain unit generally weighs about 150,000 daltons. Each L chain is linked to the H chain by one disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H-chain isotype. The H and L chains each also have regularly spaced interchain disulfide bridges. Each H chain has a variable domain (V H ) at the N-terminus, followed by three constant domains (C H ) for each of the α and γ chains, and four C H domains for the μ and ε isotypes. Each L chain has a variable domain (V L ) at the N-terminus, followed by a constant domain at the opposite end. V L is V​H is consistent with C L is consistent with the first constant domain of the heavy chain (C H 1). Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. V H and V L pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, pages 71 and chapter 6. L chains derived from any vertebrate species can be assigned to one of two distinct types called kappa and lambda based on the amino acid sequence of their constant domains. Their heavy chain constant domains (C H ) immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further classified into subclasses based on relatively minor differences in C H 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 the heavy chain but lacks the light chain normally found in a four-chain antibody. Camelids (such as camels, llamas, or alpacas) are known to produce HCAb.

[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 the 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 H". Some V H Hs may also be known as nanobodies. Camelid sdAbs are one of the smallest known antigen-binding antibody fragments (see, for example, Hamers-Casterman et al., Nature 363:446-8 (1993), Greenberg et al., Nature 374:168-73 (1995), Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). A basic V H H has the following structure from the N-terminus to the 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 the components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminating components of its production environment, such as those due to recombinant transfected cells, are materials that would typically interfere with the study, diagnostic, or therapeutic use of the antibody, and these can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide is purified to (1) greater than 95% by weight of the antibody, in some embodiments greater than 99% by weight, as determined, for example, by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the 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 staining. An isolated antibody includes antibodies that are present in recombinant cells because at least one component of the antibody's natural environment is absent. However, typically, an isolated polypeptide or antibody is prepared by at least one purification step. An isolated polypeptide 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 domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains are referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding site. Camelid species antibodies consisting of only heavy chains have a single heavy chain variable region, which is referred to as "VHH". Thus, VHH is a special type of VH. H H L L H HH H HH H L

[0045] The term "variable" refers to the fact that certain segments of the variable domain have sequences that vary significantly by antibody. 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 domain. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in the variable domains of both the light and heavy chains. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each adopt a beta-sheet conformation that is connected by, and in some cases forms part of the loops of, three HVRs and contains four FR regions that together adopt a beta-sheet conformation that is held together in close proximity by the FR regions. 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 the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains do not directly participate in binding of the antibody to antigen but exhibit various effector functions such as involvement of the antibody in antibody-dependent cell cytotoxicity.

[0046] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic site. In contrast to polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are free of contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present application may be produced, for example, by the hybridoma method (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, 2 nd(ed. 1988), Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display techniques (see, e.g., 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 the production of human or human-like antibodies in animals having a human immunoglobulin locus or a part or all of a gene encoding a human immunoglobulin sequence (see, e.g., WO1998 / 24893, WO1996 / 34096, WO1996 / 33735, WO1991 / 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 It can be produced by various techniques including, for example, 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 a 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, in contrast to antibody fragments. Specifically, it includes those having a heavy chain and a light chain that contain the full-length four-chain antibody Fc region. An antibody consisting of only a full-length heavy chain contains the heavy chain (V H H, etc.) and the Fc region. The constant domain can be the constant domain of the native sequence (e.g., the constant domain of the human native sequence) or an amino acid sequence variant thereof. In some cases, an intact antibody can have one or more effector functions.

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

[1995] ); single-chain antibody molecules; single-domain antibodies (V Hsuch as H, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody yields two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragment, which is designated to reflect its ability to crystallize readily. A Fab fragment consists of the variable region domain (V H ) of the H chain, as well as the first constant domain (C H 1) of one heavy chain. 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 corresponds approximately to two Fab fragments with different antigen-binding activities disulfide-bonded together and can still cross-link antigens. A Fab’ fragment differs from a Fab fragment in having several additional residues at the carboxy terminus of the C H 1 domain, including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for a Fab’ in which the cysteine residue(s) of the constant domain has a free thiol group. An F(ab’)2 antibody fragment was originally produced as a pair of Fab’ fragments having hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

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

[0051] ​"Fv" is the smallest antibody fragment that contains a complete antigen recognition site and antigen binding site. This fragment consists of a dimer in which one heavy chain variable region domain and one light chain variable region domain are tightly bound non-covalently. The folding of these two domains gives rise to six hypervariable loops (three loops each from the H chain and L chain) that provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of the Fv that contains only three HVRs specific for the antigen), which has a lower affinity than the full binding site, has the ability to recognize and bind the antigen.

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

[0053] The "functional fragment" of the antibodies described herein includes a portion of the intact antibody, which generally includes the antigen binding region or variable region of the intact antibody, or the Fc region of an antibody that retains or has a 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 small antibody fragment prepared by constructing an sFv fragment (see the previous paragraph) using short linkers (about 5-10 residues) between the V and V domains to achieve inter-chain rather than intra-chain V domain pairing, thereby obtaining a bivalent fragment, i.e., a fragment having two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossed" sFv fragments where the V and V domains are present on different polypeptide chains. Diabodies are described, for example, in European Patent No. 404,097, International Publication No. WO93 / 11161, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). H and V L domains to construct an sFv fragment (see the previous paragraph). A bispecific diabody is a heterodimer of two "crossed" sFv fragments where the V and V domains are present on different polypeptide chains. Diabodies are described, for example, in European Patent No. 404,097, International Publication No. WO93 / 11161, Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). H and V L domains are present on different polypeptide chains. Diabodies are described, for example, in European Patent No. 404,097, International Publication No. WO93 / 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 the corresponding sequence in an antibody 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 the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, provided that they exhibit the desired biological activity (U.S. Patent 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, where the antigen-binding region of this antibody is derived from an antibody produced, for example, by immunizing cynomolgus monkeys with the antigen of interest. When used herein, "humanized antibody" is used as a subset of "chimeric antibody".

[0056] "Humanized" forms of non-human (e.g., camelid) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVRs (defined below) have been replaced with residues from the HVRs of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, that have the desired specificity, affinity, and / or activity. In some instances, framework ("FR") residues of the human immunoglobulin have been replaced with the corresponding non-human residues. Additionally, a humanized antibody may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications can be made to further improve the performance of the antibody, such as its binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin sequence and all or substantially all of the FR regions correspond to those of the human immunoglobulin sequence, but the FR regions may include one or more individual FR residue substitutions that improve the performance of the antibody, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs is typically six or fewer in the heavy chain and three or fewer in the light chain. A humanized antibody may also optionally include at least a portion of the immunoglobulin constant region (Fc), typically at least a portion 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), as well as 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 the amino acid sequence 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 human antibody clearly excludes humanized antibodies that contain 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). The 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. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared, for example, by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge but has an inactivated endogenous locus (e.g., see U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). For example, for human antibodies produced by human B cell hybridoma technology, see also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0058] As used herein, the terms "hypervariable region", "HVR", or "HV" refer to regions of an antibody variable domain in which the sequences are hypervariable and / or form structurally defined loops. Generally, sdAbs contain three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 exhibits the highest diversity among the three HVRs and is thought to play a unique role in conferring excellent specificity to the antibody. 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 "complementary determining region" or "CDR" is used to refer to hypervariable regions as defined by the Kabat method. 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 descriptions are used and included herein. Kabat Complementary 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 positions 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 the analysis of available complex crystal structures. The residues of each of these HVRs are described in Table 1 below.

Table 1

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

[0062] sdAb (V H H, etc.) amino acid residues are, in the paper by Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23;240(1 - 2):185 - 195, V from camelids 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 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, it is well known in the art that 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 be unoccupied in the actual sequence or the actual sequence may contain more amino acid residues than permitted by the Kabat numbering).

[0063] The terms "Kabat - like variable domain residue numbering" or "Kabat - like amino acid position numbering", and variations thereof, refer to the numbering system used in the Kabat et al. (supra) for the heavy - chain variable domain or the light - chain variable domain in the construction of antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or HVR of the variable domain. For example, the heavy - chain variable domain may contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2, and residues inserted 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 alignment in the homologous regions between the sequence of the antibody and the sequence numbered by "standard" Kabat.

[0064] Unless otherwise indicated herein, the numbering of residues within an immunoglobulin heavy chain is that of the EU index as in the above - mentioned Kabat et al. "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody.

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

[0066] "Human consensus framework" or "acceptor human framework" is a framework representing the amino acid residues that most commonly occur in the selection of human immunoglobulin V L or V H framework sequences. Generally, the selection of human immunoglobulin V L or V H sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is from Kabat et al., Sequences of Proteins of Immunological Interest, 5 thIs a subgroup such as that in Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). As an example includes those related to V L and the subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV such as in Kabat et al. above. Further, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III such as in Kabat et al. Alternatively, the human consensus framework can be derived from the above, for example, when human framework residues are selected based on their homology to the donor framework sequence by aligning a donor framework sequence with a collection of various human framework sequences. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or it may contain existing amino acid sequence variations. In some embodiments, the number of existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0067] For example, "amino acid modification" at a designated position in the Fc region refers to substitution or deletion of the designated residue, or insertion of at least one amino acid residue adjacent to the designated residue. "Adjacent" insertion to the designated residue means insertion within 1 to 2 residues thereof. The insertion can be on the N-terminal side or the C-terminal side of the designated residue. Preferred amino acid modifications herein are substitutions.

[0068] An "affinity matured" antibody has one or more changes in one or more of its HVRS, and these changes result in an improvement in the affinity of the antibody for the antigen, compared to the parental antibody that does not have those change(s). In some embodiments, the affinity matured antibody has a 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) describes affinity maturation by V H and V L domain shuffling. Random mutagenesis of HVRs and / or framework residues is 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 binds", "specifically recognizes", 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 can be an epitope) binds to this target with a higher affinity, binding strength, more readily, and / or for a longer duration than it binds to other targets. In some embodiments, the degree to which an antigen-binding protein (such as a CAR or sdAb) binds to an irrelevant target is, for example, less than about 10% of the antigen-binding protein (such as a CAR or sdAb) as measured 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. In some embodiments, the antigen-binding protein (such as a CAR or sdAb) specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding may 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 particular epitope of an antigen. For example, a natural antibody is 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, and at least two of these 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) having one or more binding sites that each bind to the same antigen.

[0071] The term "valency", as used herein, indicates 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" indicate the presence of two, three, four, five, and six binding sites, respectively, in an antigen-binding protein (such as a CAR or sdAb).

[0072] The "effector function" of an antibody refers to the biological activities attributable to the Fc region of the antibody (either the native sequence Fc region or an amino acid sequence variant Fc region) and varies depending on the isotype of the antibody. 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. "Reduced or minimized" antibody effector function indicates at least a 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) reduction from a wild-type or unmodified antibody. The determination of antibody effector function is readily determinable and measurable by one of ordinary skill in the art. In preferred embodiments, the antibody effector functions of complement binding, complement-dependent cytotoxicity, and antibody-dependent cell-mediated cytotoxicity are affected. In some embodiments, the effector function is eliminated through mutations in the constant region that eliminate glycosylation, such as "effectorless mutations". In one aspect, the effectorless mutation is C HIt is the N297A or DANA mutation (D265A + N297A) in the 2 domain. Shields et al., J. Biol. Chem. 276(9):6591 - 6604(2001). Alternatively, 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 a non - glycosylating host cell (e.g., E. coli.), or in a host cell that results in an altered glycosylation pattern that is ineffective or less effective in 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. The antibody "arms" the cytotoxic cells and is necessary for killing target cells by this mechanism. While NK cells, which are primary cells for mediating ADCC, express only FcγRIII, monocytes express FcγRI, FcγRII, and FcγRIII. 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 evaluate the ADCC activity of a molecule of interest, U.S. Patent No. 5,5 An in vitro ADCC assay such as the assay described in No. 00,362 or U.S. Pat. No. 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest may be evaluated in vivo in an animal model such as those disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0074] As used herein, the term "Fc region" is defined to include the C-terminal region of an immunoglobulin heavy chain and includes both native sequence Fc regions and variant Fc regions. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is generally defined to extend from the amino acid residue at position Cys226 or Pro230 to its 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 recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include an antibody population in which all K447 residues have been removed, an antibody population without removed K447 residues, and an antibody population having a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0075] "Binding affinity" generally refers to the total strength 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 that 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 molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high-affinity antibodies generally bind antigen more rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of this application. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

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

[0077] With respect to a peptide, polypeptide, or antibody sequence, "percent amino acid sequence identity (%)" and "homology" are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of sequence identity, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps as necessary. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum 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 endow an immune effector cell, such as a T cell, with one or more antigen specificities. 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) of a T cell and / or other receptor, a transmembrane domain, and an intracellular signaling domain. "CAR-T" refers to a T cell that expresses a CAR. "BCMA CAR" refers to a CAR that has an extracellular antigen-binding domain specific for BCMA. "Two-epitope CAR" refers to a CAR that has an extracellular binding domain 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 has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components related to the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form other than the form or setting in which they are found in nature. Thus, the isolated nucleic acid molecules are distinct from the nucleic acids encoding the polypeptides and antibodies herein that are naturally present in cells.

[0080] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences 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 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 do not have to 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 ligated. This term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of inducing 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 later reintroduced into that individual.

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

[0085] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which an exogenous nucleic acid is introduced or transferred into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic 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 synonymously and all such designations include progeny. Thus, "transfectants" and "transfected cells" include the primary subject cell and cultures derived therefrom regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or accidental mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cells are included.

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

[0088] As used herein, "treatment" or "treating" refers to an approach for obtaining a beneficial or desired result, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, the following: alleviation of one or more symptoms resulting from a disease, attenuation of the degree of the disease, stabilization of the disease (e.g., prevention or delay of disease progression), prevention or delay of the spread of the disease (e.g., metastasis), prevention or delay of recurrence of the disease, delay or deceleration of the progression of the disease, improvement of the condition, provision of remission (partial or total) of the disease, reduction of the dosage of one or more other agents required for the treatment of the disease, delay of the progression of the disease, improvement of the quality of life, and / or extension of the survival period. Reduction of the pathological consequences of cancer is also included in "treatment". The methods of the present application contemplate any one or more of these treatment modalities.

[0089] As used herein, "individual" or "subject" refers to a mammal including, but not limited to, humans, cows, horses, cats, dogs, rodents, or primates. 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, an engineered immune effector cell, or a pharmaceutical composition thereof, sufficient to treat a particular disorder, condition, or disease, e.g., to ameliorate, alleviate, reduce, 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 to prevent or delay other unwanted cell proliferation. In some embodiments, the effective amount is an amount sufficient to delay onset. In some embodiments, the effective amount is an amount sufficient to prevent or delay recurrence. The effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) decrease the number of cancer cells, (ii) decrease tumor size, (iii) prevent, retard, delay to some extent, preferably halt, cancer cell infiltration into peripheral organs, (iv) prevent (i.e., delay to some extent, preferably halt) tumor metastasis, (v) prevent tumor growth, (vi) prevent or delay the occurrence and / or recurrence of tumors, and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer.

[0091] "Adjuvant setting" refers to a clinical situation in which an individual has a history of cancer and is (although not necessarily) generally responding to therapies including, but not limited to, surgery (e.g., resection), radiation therapy, and chemotherapy. However, due to their history of solid cancer, these individuals are considered at risk of developing the disease. Treatment or administration in an "adjuvant setting" refers to subsequent treatment modalities. The degree of risk (e.g., when an individual in an adjuvant setting is considered "high risk" or "low risk") depends on several factors, most commonly the extent of the disease at the time of initial treatment. Or administration refers to subsequent treatment modalities. The degree of risk (e.g., when an individual in an adjuvant setting is considered "high risk" or "low risk") depends on several factors, most commonly the extent of the disease at the time of initial treatment.

[0092] "Neoadjuvant setting" refers to the clinical situation in which the method is performed prior to primary / causal therapy.

[0093] As used herein, "delaying" the onset of cancer means withholding, interfering with, delaying, retarding, stabilizing, and / or postponing the onset of the disease. This delay can be for different periods depending on the medical history and / or individual being treated. As will be apparent to those 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 a method that reduces the likelihood of disease onset and / or reduces the extent of the disease within a given time frame as compared to not using the method. Such a comparison is typically based on clinical studies using a statistically significant number of individuals. Cancer onset can be detected using standard methods including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset can also refer to cancer progression that may not be initially detectable and includes occurrence, recurrence, and onset.

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

[0095] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. In many cases, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; 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 nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™, or polyethylene glycol (PEG).

[0096] The intended "diluent" is, in the context of this specification, pharmaceutically acceptable (safe and non-toxic for human administration) and useful for the preparation of liquid formulations, such as those that are reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (such as phosphate buffered saline), sterile aqueous saline solutions, Ringer's solution, or dextrose solutions. In an alternative embodiment, the diluent may comprise an aqueous solution of salts and / or a buffer.

[0097] A "preservative" is a compound that may be added to the formulations herein to reduce bacterial activity This is the case. The addition of a preservative can, for example, facilitate the production of a preparation for multiple uses (multiple dosages). Examples of possible preservatives include, for example, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides where 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 in this specification is benzyl alcohol.

[0098] A "stable" formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity during storage. A variety of 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, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29 - 90 (1993). Stability can be measured over a selected period of time at a selected temperature. For rapid screening, the formulation can be stored at 40°C for 2 weeks to 1 month, at which point stability is measured. When the formulation is stored at 2 - 8°C, generally, the formulation should be stable at 30°C or 40°C for at least 1 month and / or stable at 2 - 8°C for at least 2 years. When the 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. Reconstituted formulations are suitable for administration (e.g., subcutaneous administration) to a patient being treated with the protein of interest and, in some embodiments of the present application, can be suitable for parenteral or intravenous administration.

[0100] An "isotonic" formulation 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 having an osmotic pressure lower than that of human blood. Similarly, the term "hypertonic" is used to describe a formulation having an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using a vapor pressure or a freezing point 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 the embodiments of the present application described herein include the embodiments "consisting of" and / or "essentially consisting of".

[0102] References to "about" values or parameters herein include (and describe) variations that target the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

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

[0104] The term "about X - Y" as used herein 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 antibody One aspect of the present application provides an isolated single-domain antibody that specifically binds to BCMA, such as human BCMA (referred to herein as "anti-BCMA sdAb"). In some embodiments, the anti-BCMA sdAb modulates BCMA activity. In some embodiments, the anti-BCMA sdAb is an antagonist antibody. The anti-BCMA An antigen-binding fragment derived from any one of the sdAbs, and an antigen-binding protein comprising any one of the anti-BCMA sdAbs described herein are further provided. 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, namely TNFRSF17 (Thompson et al., J. Exp. Medicine, 192(1):129-135, 2000). Human BCMA is almost invariably expressed in 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 a ligand including proliferation (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 sdAb can be used in combination with cell immunotherapy using CAR-T cells to enhance, for example, the cytotoxic effect against tumor cells.

[0108] In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 115. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 116. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 117. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 118. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 119. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 120. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 121. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 122. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 123. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 124. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 125. In some embodiments, sequence number Anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of No. 126. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 127. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 128. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 129. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 130. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 131. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 132. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 133. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 134. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 135. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 136. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 137. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 138. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 139. In some embodiments, anti-BCMA sdAbs are provided that contain one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 140.In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 141. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 142. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 143. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 144. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 145. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 146. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 147. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 148. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 149. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 150. In some embodiments, anti-BCMA sdAbs are provided that include one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 151. In some embodiments, anti-BCMA sdAbs are provided that include 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 includes a receptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0109] In some embodiments, (a) CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 1 to 38, (b) CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 39 to 76, and (c) at least one, at least two, or all three CDRs selected from CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 77 to 114 are provided. In some embodiments, the anti-BCMA sdAb is of camelid origin. 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.

[0110] In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein: (a) CDR1 has 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) CDR2 has 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) CDR3 has 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. In some embodiments, a CDR having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions as compared to the reference sequence, but the anti-BCMA sdAb comprising such sequence retains the ability to bind to BCMA. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein: (a) CDR1 has about 1, 2, 3, or 4 amino acid substitutions (e.g., conservative substitutions), insertions, or deletions with respect to an amino acid sequence selected from SEQ ID NOs: 1-38; (b) CDR2 has about 1, 2, 3, or 4 amino acid substitutions (e.g., conservative substitutions), insertions, or deletions with respect to an amino acid sequence selected from SEQ ID NOs: 39-76; and (c) CDR3 has about 1, 2, 3, or 4 amino acid substitutions (e.g., conservative substitutions), insertions, or deletions with respect 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, such as a human immunoglobulin framework or a human consensus framework.

[0111] In some embodiments, provided is an anti-BCMA sdAb comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, provided is an anti-BCMA sdAb comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 2, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, provided is an anti-BCMA sdAb comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 3, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, provided is an anti-BCMA sdAb comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, provided is an anti-BCMA sdAb comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 5, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 81. In some embodiments, (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (b) An anti-BCMA sdAb comprising three CDRs is provided, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 83. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 10, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 11, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, an anti-BCMA An sdAb is provided. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 13, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 14, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 15, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 17, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 18, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 94. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 19, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 57, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 20, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, an anti-BCMA... An sdAb is provided. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, the three CDRs comprising (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, the three CDRs comprising (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 22, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 23, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and (c) the amino acid sequence of SEQ ID NO: 99 Provided are anti-BCMA sdAbs comprising three CDRs, including a CDR3. In some embodiments, provided are anti-BCMA sdAbs comprising three CDRs, including (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, provided are anti-BCMA sdAbs comprising three CDRs, including (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, provided are anti-BCMA sdAbs comprising three CDRs, including (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, provided are anti-BCMA sdAbs comprising 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, provided are anti-BCMA sdAbs comprising three CDRs, including (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, provided are anti-BCMA sdAbs comprising three CDRs, including (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, provided are anti-BCMA sdAbs comprising three CDRs, including (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, provided are anti-BCMA sdAbs comprising three CDRs, including (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 comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 32, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 108. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 33, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 109. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 34, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 110. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 35, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 73, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 111. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 36, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 112. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 37, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 75, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, an anti-BCMA sdAb comprising three CDRs is provided, wherein the three CDRs comprise: (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 38, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and (c) 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, such as a human immunoglobulin framework or a human consensus framework.

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

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

[0114] In some embodiments, the functional epitope can be mapped by combinatorial alanine scanning. In this process, a combinatorial alanine scanning strategy can be used to identify the amino acids in the BCMA protein required for interaction with the anti-BCMA sdAb. In some embodiments, the epitope is a conformation, and the epitope can be identified using the crystal structure of the anti-BCMA sdAb bound to BCMA. 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 to 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 to 394.

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

[0116] In some embodiments, the competitive assay can be used to identify monoclonal antibodies that compete with the anti-BCMA sdAbs described herein for binding to BCMA. A competitive assay can be used to determine whether two antibodies bind to the same epitope by recognizing that one antibody competitively inhibits the binding of the other antibody to the same or sterically overlapping epitope or antigen of another antibody. 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 competitive 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, N.Y.). Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). 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, the antibodies that compete with the anti-BCMA sdAbs described herein are camelid, chimeric, humanized, or human antibodies. In some embodiments, the present application provides antibodies that compete with the camelid, chimeric, humanized, or human anti-BCMA sdAbs described herein. These are included, but not limited to. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). 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, the antibodies that compete with the anti-BCMA sdAbs described herein are camelid, chimeric, humanized, or human antibodies. In some embodiments, the present application provides antibodies that compete with the camelid, chimeric, humanized, or human anti-BCMA sdAbs described herein.

[0117] In some embodiments, an anti-BCMA antibody or antigen-binding protein conjugate comprising any one of the anti-BCMA sdAbs described above is provided. In some embodiments, the anti-BCMA antibody is a monoclonal antibody comprising a camelid, chimeric, humanized, or human antibody. In some embodiments, the anti-BCMA antibody is an antibody fragment, e.g., a V H H fragment. 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 one or combination of the features described in Sections 1-7 of "Antibody Characteristics" below.

[0119] In some embodiments, an isolated nucleic acid encoding any one of the anti-BCMA antibodies (such as 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, a method of making an anti-BCMA antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the anti-BCMA antibody provided above under conditions suitable for the expression of the anti-BCMA antibody and optionally recovering the anti-BCMA antibody from the host cell (or host cell culture medium).

[0120] Characteristics of Antibodies 1. Antibody Affinity In some embodiments, the anti-BCMA antibodies provided herein have a dissociation constant (Kd) 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 to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0121] In some embodiments, the Kd is measured by a radioisotope-labeled antigen binding assay (RIA) performed using the Fab version or VH fragment of the antibody of interest and its antigen, as described by the following assay. For example, the solution binding affinity of the Fab for the antibody is determined by equilibrating the Fab with the lowest concentration of ( H I) labeled antigen in the presence of a series of titrations of unlabeled antigen and then capturing the bound antibody on a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol 125 . Biol. 293:865-881 (1999)). . 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 about 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 (about 0.2 μM) using 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 injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For the measurement of the reaction rate, 2-fold serial dilutions of the Fab or V H H of the antibody of interest (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C. The association rate (kon) and dissociation rate (koff) are calculated by fitting the association sensorgram and dissociation sensorgram simultaneously using a simple 1:1 Langmuir binding model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on rate is 10 6 M -1 s -1If the on-rate exceeds 100%, it can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band pass) 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 with a spectrometer such as a spectrophotometer equipped with a 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. Antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, V H H, 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 scFv, 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-life, see U.S. Patent No. 5,869,046.

[0124] A diabody is an antibody fragment having two antigen-binding sites that can be bivalent or bispecific. See, for example, EP404,097, WO1993 / 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 tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

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

[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. Patent No. 4,816,567 and Morrison et 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 changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0127] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody includes one or more variable domains in which the HVRs, such as CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody will optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0128] Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further 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 remodeling”), 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 the “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 specific subgroups of light chain variable regions 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 (somatic 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., J. Biol. Chem. 272:10678-1068 4 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)), but are not limited thereto.

[0130] In some embodiments, the sdAb is modified, e.g., humanized, to reduce its immunogenicity against heterologous species while not reducing the native affinity of the domain for the antigen. For example, the amino acid residues of the antibody variable domain (V H H) of a camelid antibody can be determined, and for example, one or more of the camelid amino acids within the framework region 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 antigen-binding ability of the polypeptide resulting from humanization is not significantly affected. Humanization of a camelid sdAb requires the introduction and mutagenesis of a limited amount of amino acids within a single polypeptide chain. This is in contrast to the humanization of scFv, Fab’, (Fab’)2, and IgG, which requires the introduction of amino acid changes into two chains (light and heavy chains) and the preservation of the assembly of both chains.

[0131] V H Single-domain antibodies containing an H domain can be humanized to have a human-like sequence. In some embodiments, the V used herein H The FR regions of the H domain have at least about 50%, 60%, 70%, 80%, 90%, 95%, or more amino acid sequence homology to the human V H framework region. One exemplary class of humanized V H H domains is where the V H H has, according to Kabat numbering, an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45 (e.g., L45, etc.) and tryptophan at position 103. Thus, polypeptides belonging to this class show high amino acid sequence homology to the human V H framework region, and the polypeptide can be administered directly to humans without expecting an unwanted immune response therefrom and without imposing a burden on further humanization.

[0132] Another exemplary class of humanized camelid sdAbs is described in WO03 / 035694 and contains hydrophobic FR2 residues that compensate for this loss of hydrophilicity by substituting the conserved tryptophan residue present in the V H derived from a double-stranded antibody with a charged arginine residue at position 103. Thus, peptides belonging to these two classes show high amino acid sequence homology to the human V H framework region, and the peptide can be administered directly to humans without expecting an unwanted immune response therefrom and without imposing a burden on further humanization.

[0133] 4. Human Antibodies In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of 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 an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigen administration. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or is present extrachromosomally, or is randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For an overview 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 the XENOMOUSE™ technology), U.S. Patent No. 5,770,429 (describing the HUMAB® technology), U.S. Patent No. 7,041,870 (describing the K-M MOUSE® technology), and U.S. Patent Application Publication No. US2007 / 0061900 (describing the VELOCIMOUSE® technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0135] Human antibodies can also be produced by methods based on hybridomas. Human myelomas and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, 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 in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (triooma 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 a human-derived phage display library. Such 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 target HOne technique for obtaining the VH array is to suitably immunize a transgenic mammal capable of expressing a heavy-chain antibody (i.e., to elicit an immune response and / or a heavy-chain antibody directed against said antigen or target), and then, from said transgenic mammal containing the VH H array (nucleic acid sequence encoding it), obtain a suitable biological sample (such as a blood sample, serum sample, or B cell sample, etc.), and then, starting from said sample, use any suitable technique known per se (such as any of the methods described herein or hybridoma technology, etc.) to generate a VH H array directed against said antigen or target. For example, for this purpose, the heavy-chain antibody-expressing mice described in WO02 / 085945, WO04 / 049794, and WO06 / 008548, as well as Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10;103(41):15130-5, and further methods and techniques can be used. For example, such heavy-chain antibody-expressing mice can express heavy-chain antibodies having (single) variable domains derived from natural sources (such as human (single) variable domains, camelid (single) variable domains, or shark (single) variable domains), as well as any suitable (single) variable domains such as, for example, synthetic or semi-synthetic (single) variable domains.

[0138] 5. Antibodies from libraries The antibodies of the present application can be isolated by screening a combinatorial library for antibodies having the desired activity(ies). For example, various methods for generating a phage display library and screening such library for antibodies having the desired binding properties are known in the art. Such methods are described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human is outlined in Press, Totowa, NJ, 2001), for example, McCafferty et al., Nature 348:552-554, Clackson et 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., 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., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing sdAb libraries are described, see, for example, U.S. Patent No. 7371849.

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

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

[0141] 6. Multispecific Antibodies In some embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is an antibody that has binding specificity 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, the bispecific antibody 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 a cytotoxic agent 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 making multispecific antibodies include recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (Milstein and Cuello, Nature 305:537 (1983)), WO93 / 08829, and Traunecker See, e.g., et al., EMBO J. 10:3655 (1991), and the "knob-in-hole" maneuver (see, e.g., U.S. Patent No. 5,731,168), but not limited thereto. Multispecific antibodies can be made by engineering the electrostatic steering effect to create antibody Fc-heterodimer molecules (WO2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), making bispecific antibody fragments using "diabody" technology (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., 152:5368 (1994)), as well as preparing trispecific antibodies (see, e.g., Tutt et al. J. Immunol. 147:60 (1991), and making polypeptides comprising 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 having 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 deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0144] a) Substitution, insertion, and deletion variants In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 3. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 3 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 product screened for the desired activity, such as retention / improvement of antigen binding, reduction of immunogenicity, or improvement of 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 hydrophilic: 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 would involve the exchange of a member of one of these classes with another class.

[0147] Certain types of substitutional variants involve 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 have a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have certain biological properties of the parent antibody that are substantially retained. Exemplary substitutional variants are, for example, affinity matured antibodies that can be conveniently generated using affinity maturation techniques based on phage display such as those described herein. Briefly, one or more HVR residues are mutated, the mutant antibodies are displayed on phage, and screened for a particular biological activity (e.g., binding affinity).

[0148] Modifications (e.g., substitutions) may be made, for example, within the HVRs to improve antibody affinity. Such modifications may be made to HVR "hot spots", i.e., residues encoded by codons that mutate frequently during the somatic maturation process (e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or to the SDR (a-CDR), and the resulting variant V H or V LIt is tested for binding affinity. Affinity maturation by construction of a secondary library and subsequent rescreening therefrom is 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)). In some embodiments of affinity maturation, 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. The library is then screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In many cases, CDR-H3 and CDR-L3 are specifically targeted.

[0149] In some embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such modifications do not substantially reduce the ability of the antibody to bind its antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications can be within HVR “hot spots” or outside the CDRs. The variant V H H sequences provided above, in some embodiments, each HVR is either unmodified or has one, two, or three or fewer amino acid substitutions.

[0150] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is referred to as "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 by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with its antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the first substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted as candidates for substitution or can be excluded. Mutant forms can be screened to determine whether they have the desired properties.

[0151] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions having lengths in the range of polypeptides containing from 1 residue to over 100 residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertional mutant forms of the antibody molecule include fusions of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody at the N-terminal or C-terminal end of the antibody.

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

[0153] If the antibody contains an Fc region, the carbohydrates bound thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally bound by 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 can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the present application can be performed to create antibody variants having certain improved properties.

[0154] In some embodiments, antibody variants are provided that have a carbohydrate structure lacking fucose (either directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody can be 1% - 80%, 1% - 65%, 5% - 65%, or 20% - 40%. The amount of fucose is determined, for example, as described in WO2008 / 077546, by calculating the average amount of fucose within the sugar chain at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 within the Fc region (EU numbering of Fc region residues), but Asn297 may also be located between positions 294 - 300, i.e., approximately ±3 amino acids upstream or downstream from position 297, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, 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. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. B iophys.249:533-545(1986), US Patent Application No. US2003 / 0157108 A1 (Presta, L, and WO2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines such as knockout cells of the alpha-1,6-fucosyltransferase gene, FUT8, CHO cells (for example, see Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107).

[0155] For example, there is further provided an antibody variant having a bisected oligosaccharide in which the bisected oligosaccharide in which the branched oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such an antibody variant may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), US Patent No. 6,602,684 (Umana et al.), and US2005 / 0123546 (Umana et al.). There is also provided an antibody variant having at least one galactose residue in the oligosaccharide bound to the Fc region. Such an antibody variant 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 variant In some embodiments, one or more amino acid modifications are introduced into the Fc region of the antibody provided herein, whereby an Fc region variant can be generated. The Fc region variant can include a human Fc region sequence (for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (for example, substitution) at one or more amino acid positions.

[0157] In some embodiments, the present application contemplates antibody variants that possess some but not all effector functions, such that while the in vivo half-life of the antibody is important, the particular effector functions (such as complement and ADCC) are unnecessary or detrimental for the intended use. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / abrogation of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. While NK cells, which are primary cells for mediating ADCC, express only Fc(RIII, monocytes express Fc(RI, Fc(RII, and Fc(RIII. FcR expression in 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 for evaluating the ADCC activity of the molecule of interest are described in U.S. Patent No. 5,500,362 (see, for example, 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 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, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be determined in vivo, for example, as described by Clynes et It can be evaluated in the animal model disclosed in al.Proc.Nat’l Acad.Sci.USA 95:652-656(1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q, thereby lacking CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163(1996), Cragg, M.S.et al., Blood 101:1045-1052(2003), and Cragg, M.S.and M.J.Glennie, Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, S.B.et al., Int’l.Immunol.18(12):1759-1769(2006)).

[0158] Examples of antibodies with reduced effector function include antibodies having 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 mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, for example, the so-called "DANA" Fc mutant having substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0159] Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J.Biol.Chem.9(2):6591-6604(2001).)

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

[0161] In some embodiments, for example, as described in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000), the modifications introduced in the Fc region result in modified (i.e., either improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC).

[0162] Antibodies having improved binding to the neonatal Fc receptor (FcRn) involved in the prolongation of half-life and 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.). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the 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, for example, a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0163] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351.

[0164] d) Cysteine-engineered antibody variants In some embodiments, it may be desirable to generate a cysteine-engineered antibody, e.g., a “thioMab,” in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By replacing these residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody, which can be used to conjugate the antibody to other moieties, e.g., a drug moiety or a linker-drug moiety, to generate an immunoconjugate as further described herein. In some embodiments, any one or more of the following residues may be replaced with cysteine: A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. The cysteine-engineered antibody 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 can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatizing the antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may be advantageous during production due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is to be used therapeutically under defined conditions, etc.

[0166] In some embodiments, conjugates of antibodies and non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In some embodiments, the non-proteinaceous 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 non-proteinaceous moiety to a temperature that kills cells proximal to the antibody-non-proteinaceous moiety without damaging normal cells.

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

[0168] Methods for preparing sdAbs are described. See, for example, Els Pardon et al, Nature Protocol, 2014;9(3):674. Single domain antibodies (V H H, etc.) can be obtained using methods known in the art, for example, by immunizing camelid species (such as camels or llamas) and then obtaining hybridomas, 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 the recombinant production of sdAbs, the nucleic acid encoding the sdAb is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. The DNA encoding the sdAb can be easily isolated and sequenced using conventional techniques (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell used. Generally, preferred host cells are either of prokaryotic origin or of eukaryotic (generally mammalian) origin.

[0170] 1. Polyclonal antibodies Polyclonal antibodies are generally produced 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, SOCl2, or R 1N=C=NR (wherein R and R 1 are independently lower alkyl groups) can be used to conjugate a 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. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol can be selected by those skilled in the art without undue experimentation.

[0171] Animals are immunized against an antigen, immunogenic conjugate, or derivative by, for example, combining 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boost-immunized with 1 / 5 to 1 / 10 of the original amount of the peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the sera are assayed for antibody titers. The animals are boost-immunized until the titer reaches a plateau. The conjugate can also be prepared in recombinant cell culture as a protein fusion. Also, aggregating agents such as alum are 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 comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in trace amounts. Thus, the modifier "monoclonal" indicates the characteristic of the antibody that it is not a mixture of discrete antibodies.

[0173] For example, monoclonal antibodies can be prepared initially using the hybridoma method described by Kohler et al., Nature, 256:495 (1975), or can be prepared 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 produce, or be capable of producing, lymphocytes that will produce antibodies that specifically bind to the protein used for immunization. Alternatively, the 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 and Practice, pp. 59-103 (Academic Press, 1986).

[0175] The immunizing agent typically includes the antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes ("PBL") are used when human origin cells are desired, or spleen cells or lymph node cells are used when non-human mammalian origin is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. Goding, Monoclonal 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. Usually, rat or mouse myeloma cell lines are 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, when the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that block 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 media such as HAT medium. Among these, those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, as well as mouse myeloma strains such as the SP-2 cells (and its derivatives, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Va., USA, are preferred. Human myelomas 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 hybridoma cells grow is assayed for the production of monoclonal antibodies directed against an antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

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

[0180] Hybridoma cells that produce antibodies having the desired specificity, affinity, and / or activity After being identified, the clones are subcloned by limiting dilution technique and can be 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 can grow in vivo as tumors in mammals.

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

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

[0183] In a further embodiment, the antibody can be isolated from an antibody phage library 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) each describe the isolation of murine and human antibodies using phage libraries. Further reports describe the generation 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)). Thus, these techniques are viable alternatives to the conventional monoclonal antibody hybridoma technology for isolating monoclonal antibodies.

[0184] DNA can be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently conjugating all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of the antibody, or they are substituted for the variable domains of one antigen-binding site of the antibody to create a chimeric bivalent antibody that includes one antigen-binding site having specificity for an antigen and another antigen-binding site having 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 the recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chains are generally cleaved at any point within the Fc region so as to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues can be replaced with another amino acid residue or deleted so as to prevent cross-linking. In vitro methods are also suitable for the preparation of monovalent antibodies. The digestion of antibodies to produce their fragments, specifically Fab fragments, can be achieved using routine techniques known in the art.

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

[0187] 3. Recombinant production in prokaryotic cells a) Vector construction The polynucleotide sequences encoding the antibodies of the present application can be obtained using standard recombinant techniques. The desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells such as hybridoma cells. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the sequences encoding the polypeptides are inserted into recombinant vectors capable of replicating and expressing the heterologous polynucleotides in a prokaryotic host. Many vectors are available and can be used in the present invention that are known in the art. The selection of an appropriate vector depends mainly on the size of the nucleic acid inserted into the vector and the particular 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 particular host cell in which it is present. 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, a heterologous nucleic acid insert, and a transcription termination sequence.

[0188] Generally, plasmid vectors containing a replicon and control sequences derived from a species compatible with the host cell are used in connection with these hosts. This vector usually has a replication site and a marking sequence capable of providing phenotypic selection in the transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from the E. coli species. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet) and thus provides an easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain or can be modified to contain promoters that can be used by the microorganism for the 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] In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors in relation to these hosts. For example, bacteriophages such as GEM (trademark)-11 can be utilized in constructing recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0190] The expression vectors of the present application may include two or more promoter-cistron pairs each encoding a polypeptide component. A promoter is an untranslated control sequence located upstream (5′) of the cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes, inducible promoters and constitutive promoters. An inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in culture conditions, such as the presence or absence of nutrients, or changes in temperature.

[0191] Numerous promoters recognized by various potential host cells are well known. The selected promoter can 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 natural promoter sequences and many heterologous promoters can be used to induce amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally result in greater transcription and higher yields of the expressed target gene compared to the natural target polypeptide promoter.

[0192] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the -lactamase and lactose promoter systems, 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, etc.) are also suitable. Their nucleic acid sequences are publicly available, thereby enabling those skilled in the art to operably link them to the 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 within the recombinant vector contains a secretory signal sequence component that induces translocation of the expressed polypeptide across the membrane. Generally, the signal sequence can be a component of the vector or part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be recognized and processed by the host cell (i.e., cleaved by signal peptidase). For prokaryotic host cells that do not recognize or process the signal sequence native to the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, 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 the 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, such as the V of the first antigen-binding portion optionally fused to a second antigen-binding portion HA polypeptide encoding a domain and the V of a first antigen-binding portion optionally fused to a second antigen-binding portion L A polypeptide encoding a domain, etc. is expressed, folded, and assembled to form a functional antibody in the cytoplasm. A particular host strain (e.g., E. coli trxB - strain) provides cytoplasmic conditions favorable for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

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

[0196] Preferably, a set of vectors is generated at a certain range of TIR intensities for each cistron within it. This limited set provides a comparison of the expression levels of each strand, as well as the yield of the desired protein product under various TIR intensity combinations. TIR intensity can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al., U.S. Patent No. 5,840,523. Based on the translation intensity comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the present application.

[0197] b) Prokaryotic host cells Prokaryotic host cells suitable for the expression of the antibodies of the present application include Archaebacteria and Eubacteria such as Gram-negative or Gram-positive organisms. 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 marcescens, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In some embodiments, Gram-negative cells are used. In some embodiments, E. coli cells are used as the host of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219, ATCC deposit number 27,325) and its derivatives (genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A(nmpc-fepE) degP41 kan Rincluding stock 33D3 having it) (U.S. Patent No. 5,639,635). Other stocks and their derivatives, for example, E. coli 294 (ATCC 31,446), E. coli B, E. coli 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-described bacteria having a defined genotype 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 considering the replicability of the replicon in the bacterial cell. For example, when providing a replicon using a well-known plasmid such as pBR322, pBR325, pACYC177, or pKN410, E. coli, Serratia, or Salmonella species can be suitably used as hosts.

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

[0199] c) Protein production The host cell is transformed with the above-described expression vector and cultured in a conventional nutrient medium modified to be suitable for induction of the promoter, selection of the transformant, or amplification of the gene encoding the desired sequence. Transformation means introducing DNA into a prokaryotic host so that the DNA can be replicated either as an extrachromosomal element or by a chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. In general, calcium treatment using calcium chloride is used for bacterial cells containing a substantial cell wall barrier. Another method of transformation is using polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0200] The prokaryotic cells used to produce the antibodies of the present application are known in the art and grow in a medium suitable for culturing the selected host cells. Examples of suitable media include Luria Broth (LB) containing the necessary nutrient supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for growing cells expressing the 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 as a mixture with another supplement such as a complex nitrogen source or the medium. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithiothreitol, and dithioerythritol.

[0202] Prokaryotic host cells are cultured at a suitable temperature. For E. coli growth, for example, the preferred temperature ranges 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, mainly depending 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 activation of the promoter. In one aspect of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Thus, the transformed host cells are cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263: 133-147). Various other inducers can be used according to vector constructs known in the art.

[0204] The expression antibody of the present application is secreted into the periplasm of the host cell and then recovered from there. Protein recovery typically involves disruption of the microorganism by means such as osmotic shock, sonication, or lysis in general. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein is 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 is filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further isolated and identified using generally known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0205] Alternatively, protein production is carried out in large quantities by a fermentation process. Various large-scale fed-batch fermentation techniques are available for the production of recombinant proteins. Large-scale fermentation has a capacity of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermenters use agitation impellers to distribute oxygen and nutrients, particularly glucose (preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in a fermenter 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 after the cells have grown under suitable conditions until they reach a desired density, for example, an OD of about 180 - 220, at which point the cells are in the early stationary phase. Various inducers can be used according to the above-described vector constructs known in the art. The cells can grow for a shorter period before induction. The cells are usually induced for about 12 - 50 hours, although longer or shorter induction times can also be used. 550 Once the cells reach the desired density, for example, an OD of about 180 - 220, induction of protein expression is typically initiated, at which point the cells are in the early stationary phase. Various inducers can be used according to the above-described vector constructs known in the art. The cells can grow for a shorter period before induction. The cells are usually induced for about 12 - 50 hours, although longer or shorter induction times can also be used.

[0207] To improve the production yield and quality of the antibodies of the present application, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted polypeptide, a further vector that overexpresses a chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidylprolyl cis,trans-isomerase with chaperone activity), can be used to co-transform the host prokaryotic cell. It has been demonstrated that chaperone proteins 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 the expressed heterologous protein (especially those sensitive to proteolysis), certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, the host cell strain can be modified to introduce a genetic mutation(s) in the gene encoding a known bacterial protease, 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, for example, as described in Joly et al. (1998) (see above), Georgiou et al., US Patent No. 5,264,365, Georgiou et al., US Patent No. 5,508,192, Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0209] An E. coli strain transformed with a plasmid lacking a protease and overexpressing one or more chaperone proteins can be used as a host cell in an expression system encoding the antibody of the present application.

[0210] d) Protein purification The antibodies produced herein are further purified to obtain a substantially homogeneous preparation for further assays and use. Standard protein purification methods known in the art can be used. The following techniques: fractionation on an immunoaffinity or ion exchange column, 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 are illustrative of suitable purification techniques.

[0211] In one aspect, protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody containing the Fc region of the present application. Protein A is a 41 kD cell wall protein derived from Staphylococcus aureas that binds to the Fc region of the antibody 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 silica column. In some applications, the column is coated with a reagent such as glycerol to prevent non-specific attachment of contaminating substances. The solid phase is then washed to remove contaminating substances that have bound non-specifically to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0212] 4. Recombinant production in eukaryotic cells In the case of 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 in eukaryotic hosts can also be inserts encoding a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The selected heterologous signal sequence is preferably one that is recognized and processed by the host cell (i.e., cleaved by signal peptidase). In mammalian cell expression, mammalian signal sequences, as well as viral secretion leaders, such as the herpes simplex gD signal, are available.

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

[0215] b) Origin of replication Generally, an origin of replication component is not necessary for mammalian expression vectors (the SV40 origin can typically be used only for the reason that it contains an early promoter).

[0216] c) Selectable gene component Expression and cloning vectors can contain a selectable gene, also known as a selectable marker. Typical selectable genes encode a protein that (a) confers resistance to an antibiotic or other toxin, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complements an auxotrophic deficiency, or (c) supplies an essential nutrient not available from complex media, such as the gene encoding D-alanine racemase for Bacilli.

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

[0218] Another example of a selectable marker suitable for mammalian cells is one that enables the identification of cell components for taking up nucleic acids encoding the antibodies of the present application such as DHFR, thymidine kinase, metallothionein-I and -II, preferably the primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc.

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

[0220] Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with a polypeptide-encoding DNA sequence, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in a medium containing a selective 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 operably linked to the nucleic acid encoding the desired polypeptide sequence. Substantially all eukaryotic genes have an AT-rich region located approximately 25 - 30 bases upstream from the site where transcription is initiated. Another sequence found 70 - 80 bases upstream from the transcription start of many genes is the CNCAAT region where N can be any nucleotide. The 3' end of most eukaryotes is the AATAAA sequence, which can be a signal for the 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 β-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 (S.D.) sequence that is operably linked to the DNA encoding the antibody.

[0223] Transcription of polypeptides from vectors in mammalian host cells is controlled by promoters obtained from the genomes of viruses such as, for example, polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, Rous sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter or the immunoglobulin promoter, or the heat shock promoter, 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 SV40 restriction fragments that also contain the SV40 origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as the HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications of this system are described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) regarding the 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 components Transcription of the DNA encoding the antibodies of the present application by higher eukaryotes is often increased by inserting enhancer sequences into the vector. Many enhancers sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are currently known. However, typically, enhancers derived from eukaryotic cell viruses will be used. Examples include the SV40 enhancer (bp100-270) in the latter half of the origin of replication, the cytomegalovirus immediate early promoter enhancer, the polyoma enhancer in the latter half of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297:17-18 (1982) regarding enhancer elements for activation of eukaryotic promoters. Enhancers can be spliced into the vector at the 5' or 3' position of the polypeptide coding sequence, but are preferably located at the 5' site from the promoter.

[0226] f) Transcription termination component Expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) will also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are generally available from the 5' untranslated regions of eukaryotic or viral DNA or cDNA, and sometimes from the 3' untranslated regions. These regions contain nucleotide segments transcribed as polyadenylation fragments within the untranslated portion of the polypeptide coding mRNA. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0227] g) Selection and transformation of host cells Suitable host cells for cloning or expression of DNA in vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (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), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and human liver cancer line (Hep G2).

[0228] The 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 induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences.

[0229] h) Culturing of host cells The host cells used to produce the antibodies of the present application can be cultured in various media. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. , Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO90 / 03430, WO87 / 00195, or U.S. Patent Reissue No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented, if necessary, with 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 GENTAMYCIN (trademark) drug), trace elements (defined as inorganic compounds that are usually present at a minimum concentration in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. The culture conditions such as temperature, pH, etc. are those that have been used heretofore with the host cells 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 secreted directly into the medium. When antibodies are produced intracellularly, as a first step, particulate debris (either host cells or lysis fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe procedures 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 about 30 minutes. Cell debris can be removed by centrifugation. When antibodies are secreted into the medium, the supernatant of such an expression system is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can 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. Antibodies based on human immunoglobulins containing one, two, or four heavy chains can be purified using protein A (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. 5:1567-1575 (1986)). The substrate to which the affinity ligand binds is most often agarose, although other substrates are available. Mechanically stable substrates such as controlled pore glass or poly(styrene-divinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. When the antibody is CH When containing 3 domains, Bakerbond ABXTM resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE (trademark) chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation and other techniques for protein purification are also available depending on the antibody to be recovered.

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

[0233] Immunoconjugate In some embodiments, the present application also provides an immunoconjugate comprising any of the antibodies (such as sdAb) described herein conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

[0234] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) that conjugates to one or more drugs including, but not limited to, maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. EP0425235 B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Patent 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. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see 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., 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; trichothecene; and 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 non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

[0236] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. By way of example, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu. When a radioactive conjugate 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 iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, also in this case.

[0237] ​Conjugates of antibodies and cytotoxic agents can be prepared using various bifunctional protein binders such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide 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 the conjugation of radioactive nucleotides to antibodies. See WO94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug intracellularly. For example, an acid-labile linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0238] The immunoconjugates or ADCs of this specification are clearly contemplated, but not limited to, such conjugates prepared with crosslinking reagent reagents including 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 commercially available SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0239] Methods and compositions for diagnosis 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 includes cells or tissues.

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

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

[0242] III. Chimeric Antigen Receptor One aspect of the present application is one or more single-domain antibodies (V HProvided is a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that includes, e.g., H. Any one of the anti-BCMA sdAbs described in Section II can be used in the CARs described herein. Exemplary structures of the CARs are shown in FIGS. 15A-15D.

[0243] In some embodiments, provided is a BCMA-targeting chimeric antigen receptor (CAR, referred to herein as "BCMA CAR") 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 co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory 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 of 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 the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first co-stimulatory signaling domain derived from CD28, a second co-stimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory 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, provided is a BCMA CAR 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 is one of the following: (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 2, 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: 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 containing the amino acid sequence of SEQ ID NO: 14, CDR2 containing the amino acid sequence of SEQ ID NO: 52, and CDR3 containing the amino acid sequence of SEQ ID NO: 90, (15) CDR1 containing the amino acid sequence of SEQ ID NO: 15, CDR2 containing the amino acid sequence of SEQ ID NO: 53, and CDR3 containing the amino acid sequence of SEQ ID NO: 91, (16) CDR1 containing the amino acid sequence of SEQ ID NO: 16, CDR2 containing the amino acid sequence of SEQ ID NO: 54, and CDR3 containing the amino acid sequence of SEQ ID NO: 92, (17) CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 55, and CDR3 containing the amino acid sequence of SEQ ID NO: 93, (18) CDR1 containing the amino acid sequence of SEQ ID NO: 18, CDR2 containing the amino acid sequence of SEQ ID NO: 56, and CDR3 containing the amino acid sequence of SEQ ID NO: 94, (19) CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 57, and CDR3 containing the amino acid sequence of SEQ ID NO: 95, (20) CDR1 containing the amino acid sequence of SEQ ID NO: 20, CDR2 containing the amino acid sequence of SEQ ID NO: 58, and CDR3 containing the amino acid sequence of SEQ ID NO: 96, (21) CDR1 containing the amino acid sequence of SEQ ID NO: 21, CDR2 containing the amino acid sequence of SEQ ID NO: 59, and CDR3 containing the amino acid sequence of SEQ ID NO: 97, (22) CDR1 containing the amino acid sequence of SEQ ID NO: 22, CDR2 containing the amino acid sequence of SEQ ID NO: 60, and CDR3 containing the amino acid sequence of SEQ ID NO: 98, (23) CDR1 containing the amino acid sequence of SEQ ID NO: 23, CDR2 containing the amino acid sequence of SEQ ID NO: 61, and CDR3 containing the amino acid sequence of SEQ ID NO: 99, (24) CDR1 containing the amino acid sequence of SEQ ID NO: 24, CDR2 containing the amino acid sequence of SEQ ID NO: 62, and CDR3 containing the amino acid sequence of SEQ ID NO: 100, (25) CDR1 containing the amino acid sequence of SEQ ID NO: 25, CDR2 containing the amino acid sequence of SEQ ID NO: 63, and CDR3 containing the amino acid sequence of SEQ ID NO: 101, (26) The amino acid sequence of SEQ ID NO: 26, CDR1 containing the amino acid sequence of SEQ ID NO: 64, CDR2 containing the amino acid sequence of SEQ ID NO: 102, and CDR3 containing the amino acid sequence of SEQ ID NO: 102; (27) CDR1 containing the amino acid sequence of SEQ ID NO: 27, CDR2 containing the amino acid sequence of SEQ ID NO: 65, and CDR3 containing the amino acid sequence of SEQ ID NO: 103; (28) CDR1 containing the amino acid sequence of SEQ ID NO: 28, CDR2 containing the amino acid sequence of SEQ ID NO: 66, and CDR3 containing the amino acid sequence of SEQ ID NO: 104; (29) CDR1 containing the amino acid sequence of SEQ ID NO: 29, CDR2 containing the amino acid sequence of SEQ ID NO: 67, and CDR3 containing the amino acid sequence of SEQ ID NO: 105; (30) CDR1 containing the amino acid sequence of SEQ ID NO: 30, CDR2 containing the amino acid sequence of SEQ ID NO: 68, and CDR3 containing the amino acid sequence of SEQ ID NO: 106; (31) CDR1 containing the amino acid sequence of SEQ ID NO: 31, CDR2 containing the amino acid sequence of SEQ ID NO: 69, and CDR3 containing the amino acid sequence of SEQ ID NO: 107; (32) CDR1 containing the amino acid sequence of SEQ ID NO: 32, CDR2 containing the amino acid sequence of SEQ ID NO: 70, and CDR3 containing the amino acid sequence of SEQ ID NO: 108; (33) CDR1 containing the amino acid sequence of SEQ ID NO: 33, CDR2 containing the amino acid sequence of SEQ ID NO: 71, and CDR3 containing the amino acid sequence of SEQ ID NO: 109; (34) CDR1 containing the amino acid sequence of SEQ ID NO: 34, CDR2 containing the amino acid sequence of SEQ ID NO: 72, and CDR3 containing the amino acid sequence of SEQ ID NO: 110; (35) CDR1 containing the amino acid sequence of SEQ ID NO: 35, CDR2 containing the amino acid sequence of SEQ ID NO: 73, and CDR3 containing the amino acid sequence of SEQ ID NO: 111; (36) CDR1 containing the amino acid sequence of SEQ ID NO: 36, CDR2 containing the amino acid sequence of SEQ ID NO: 74, and CDR3 containing the amino acid sequence of SEQ ID NO: 112; (37) CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 75, and CDR3 containing the amino acid sequence of SEQ ID NO: 113, or (38) CDR1 containing the amino acid sequence of SEQ ID NO: 38, CDR2 containing the amino acid sequence of SEQ ID NO: 76, and CDR3 containing 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 comprises a V comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115 to 152. H It 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 co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of 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 the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first co-stimulatory signaling domain derived from CD28, a second co-stimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory 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 that includes a polypeptide 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 the group consisting of SEQ ID NOs: 216 - 256 and 298 - 335. In some embodiments, a BCMA CAR is provided that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 - 256 and 298 - 335. A polypeptide is also provided that includes 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 that includes a polypeptide 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 the group consisting of SEQ ID NOs: 216 - 256 and 298 - 335. In some embodiments, a BCMA CAR is provided that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 - 256 and 298 - 335. A polypeptide is also provided that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 - 256 and 298 - 335.

[0246] In some embodiments, an isolated nucleic acid is provided that encodes any of the BCMA CARs provided herein. 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: 257 - 297 and 336 - 373. In some embodiments, an isolated nucleic acid is provided that includes 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 that includes any one of the nucleic acids encoding the 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. An exemplary monovalent BCMA CAR is shown in Table 4 below.

Table 4 - 1

Table 4 - 2

Table 4-3

[0247] Multivalent chimeric antigen receptor This application also provides multivalent CARs having two or more (such as any one of about 2, 3, 4, 5, 6, or more) binding moieties that specifically bind to antigens 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 include single-domain antibodies. In some embodiments, one or more of the binding moieties are derived from camelid antibodies. In some embodiments, one or more of the binding moieties are derived from four-chain antibodies. 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 human antibodies. 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, the multivalent CAR targets a single antigen and includes 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 the multivalent CAR includes more than two binding sites for at least one antigen. Binding moieties specific for the same antigen can bind to the same epitope of the antigen (i.e., "one-epitope CAR") or to different epitopes of the antigen (i.e., "multi-epitope CAR" such as two-epitope CAR or three-epitope CAR). Binding sites specific for the same antigen can include the same or different sdAbs. The binding sites specific for the same antigen can include the same or different sdAbs.

[0248] 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 one of about 2, 3, 4, 5, 6 or more) of binding moieties 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.

[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 one of about 2, 3, 4, 5, 6 or more) of 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 valency) 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., 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 moiety, such as an sdAb (including multiple sdAbs, or a first sdAb and / or a second sdAb), is camelid, chimeric, human, or humanized. In some embodiments, the binding moiety or sdAb is fused to each other via a peptide bond or a peptide linker. In some embodiments, each peptide linker has an amino acid length of about 50 or less (such as any one of about 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 includes the 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 includes a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of CD83, and combinations thereof. In some embodiments, the multivalent CAR further includes 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 includes a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide includes, from the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory 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 via synergistic binding by different antigen-binding sites or for enhancing the binding affinity or avidity for an antigen. Any of the anti-BCMA sdAbs described herein may be used as 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 shown in Table 5.

[0254] In some embodiments, there are provided multivalent CARs that target BCMA and comprise (a) an extracellular antigen-binding domain comprising a plurality (at least one of about 2, 3, 4, or more) of 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 CARs. In some embodiments, the extracellular antigen-binding domain specifically binds to a single epitope of BCMA, and these CARs are referred to herein as multivalent BCMA CARs of one epitope.

[0255] In some embodiments, there are provided multivalent BCMA CARs that comprise (a) an extracellular antigen-binding domain comprising a plurality (at least one of about 2, 3, 4, or more) of anti-BCMA sdAbs, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb comprises 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 "multiepitope multivalent CAR") is provided that includes (a) an extracellular antigen-binding domain comprising at least two (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., 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 binds 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 BCMA-binding moiety specifically binds to an epitope derived from SEQ ID NO: 391 and / or 392.

[0257] In some embodiments, a multivalent BCMA CAR is provided that includes (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 that is 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 NO: 389 and / or 390. In some embodiments, the second anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NO: 391 and / or 392.

[0258] In some embodiments, a multivalent BCMA CAR is provided that includes (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 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 VH domain 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. HIt includes an H domain.

[0259] In some embodiments, a multivalent BCMA CAR is provided that includes (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 includes 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 includes 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 includes a V H H domain. In some embodiments, the second anti-BCMA sdAb includes a V H H domain.

[0260] In some embodiments, a multivalent BCMA CAR is provided that includes (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 includes 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 includes 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 includes a V H H domain. In some embodiments, the second anti-BCMA sdAb includes a V H H domain.

[0261] In some embodiments, a multivalent BCMA CAR is provided that includes (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 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 H domain comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, the second anti-BCMA sdAb comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 132.

[0262] In some embodiments, a multivalent BCMA CAR is provided that includes (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 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 H domain comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the second anti-BCMA sdAb comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 134.

[0263] In some embodiments, a multivalent BCMA CAR is provided that includes (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 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 V H H domain comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, the second anti-BCMA sdAb comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 142.

[0264] In some embodiments, 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 to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker is of an amino acid length of about 50 or less (such as any one of about 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the intracellular signaling domain is the primary intracellular It includes a signal transduction domain. In some embodiments, the primary intracellular signal transduction domain is derived from CD3ζ. In some embodiments, the intracellular signal transduction domain includes a co-stimulatory signal transduction domain. In some embodiments, the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of CD83, and combinations thereof. In some embodiments, the multivalent BCMA CAR further includes a hinge domain (such as the 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 includes a signal peptide (such as the CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide includes, from the N-terminus to the C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signal transduction domain derived from CD137, and a primary intracellular signal transduction 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, provided is a multivalent BCMA CAR comprising a polypeptide 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 the group consisting of SEQ ID NOs: 298 to 335. In some embodiments, provided is a multivalent BCMA CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298 to 335. Also provided is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298 to 335.

[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 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: 336 - 373 is provided. 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 comprising any one of the nucleic acids encoding the multivalent BCMA CARs described above is provided. 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 receptor The present application further provides a multispecific chimeric antigen receptor that targets two or more (such as any one of about 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 include an sdAb. For example, in some embodiments, the multispecific CAR is a bispecific CAR that includes an extracellular antigen-binding domain that includes two different sdAbs that each specifically bind to an antigen. In some embodiments, the multispecific CAR is a trispecific CAR that includes an extracellular antigen-binding domain that includes three different sdAbs that each specifically bind to an antigen. binding domain.

[0268] In some embodiments, a multispecific (bispecific) chimeric antigen receptor (CAR) is provided that comprises: (a) an extracellular antigen-binding domain comprising a first single-domain antibody (sdAb) that specifically binds to BCMA and a second single-domain antibody (sdAb) that specifically binds to a second antigen (such as a tumor antigen), wherein the first antigen is different from the second antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the second antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, 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 sdAb and / or the second sdAb is camelid, chimeric, human, or humanized. In some embodiments, the first sdAb and the second sdAb are fused to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker is about 50 amino acids in length or less (such as any one of about 35, 25, 20, 15, 10, or 5 amino acids 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 co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of 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, from the N-terminus to the C-terminus, comprises a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the polypeptide, from the N-terminus to the C-terminus, comprises a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a co-stimulatory 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 the CAR described herein comprises one or more (such as 1, 2, 3, 4, 5, 6, or any one of them or more) binding moieties such as sdAbs. In some embodiments, the one or more binding moieties are antibodies or antigen-binding fragments thereof. In some embodiments, the one or more binding moieties are derived from a four-chain antibody. In some embodiments, the one or more binding moieties are derived from a camelid antibody. In some embodiments, the one or more binding moieties are derived from a human antibody. In some embodiments, the one or more binding moieties are non-antibody binding proteins, such as polypeptide ligands, or engineered proteins that bind to an antigen. The binding moieties can be directly fused to each other via a peptide bond or a peptide linker.

[0270] 1. Single-domain antibody In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising one or more sdAbs. The sdAbs can be of the same or different origin and can be of the same or different sizes . Exemplary sdAbs include heavy-chain-only antibodies (such as V H H or V NAR ) from the heavy-chain variable domain, binding molecules that are naturally lacking a light chain, single domains (VH or V L etc.), 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 sdAbs known in the art or developed by the inventors, including those described in Section II of this application, can be used to construct the CARs described herein. The sdAbs can be derived from any species, including but not limited to mice, rats, humans, camels, llamas, sharks, goats, rabbits, and cows. The single-domain antibodies contemplated herein also include naturally occurring sdAbs from species other than camels and sharks.

[0271] In some embodiments, they are derived from naturally occurring single-domain antigen-binding molecules known as heavy-chain antibodies lacking a light chain (also referred to herein as "heavy-chain-only antibodies"). Such single-domain molecules are disclosed, for example, in WO94 / 04678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, the variable domain derived from a heavy-chain molecule that is naturally lacking a light chain is designated V H herein as V H H to distinguish it from the conventional V of a four-chain immunoglobulin. Such V H H molecules can be derived from antibodies produced in camelid species, such as camels, llamas, vicunas, alpacas, and guanacos. Other species besides camelids can produce heavy-chain molecules that are naturally lacking a light chain, and such V H H is within the scope of this application.

[0272] V from camelids H H molecules are approximately 10 times smaller than IgG molecules. They are single polypeptides, are very stable, and can exhibit resistance to extreme pH and temperature conditions. Furthermore, they can exhibit resistance to protease action, which does not apply to conventional four-chain antibodies. Furthermore, V HIn vitro expression of H results in a high yield of properly folded and functional V H H. In addition, antibodies generated in camelids can recognize epitopes other than those recognized by antibodies generated in vitro, either by using antibody libraries or by immunizing mammals other than camelids (see, for example, WO9749805). Thus, a multispecific or multivalent CAR comprising one or more V H H domains can interact with a target more efficiently than a multispecific or multivalent CAR comprising an antigen-binding fragment derived from a conventional four-chain antibody. Since V H H is known to bind to "unusual" epitopes such as cavities or grooves, the affinity of a CAR comprising such V H H may be more suitable for therapeutic treatment than conventional multispecific polypeptides.

[0273] In some embodiments, the sdAb is derived from the variable region of an immunoglobulin found in cartilaginous fish. For example, the sdAb may be derived from an 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 NAR ("IgNAR") are described in WO03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0274] In some embodiments, the sdAb is recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or generated in vitro (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework region can be modified by "camelization" of specific amino acid residues within the framework region. Camelization refers to the replacement or substitution of one or more of the amino acid residues within the amino acid sequence of a (naturally occurring) V H domain from a conventional four-chain antibody with one or more of the amino acid residues occurring at the corresponding position(s) within the V H H domain of a heavy chain antibody. This is, for example, It can be carried out in a manner that is inherently known to those skilled in the art based on further descriptions in this specification. Such "camelization" substitutions preferably occur at V H -V L to form an interface and / or at the amino acid positions present at that interface and / or inserted into the so-called camelid characteristic residues defined herein (see, for example, 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 Muyldermans J. Immunol. Meth. 231:25-38, 1999).

[0275] In some embodiments, the sdAb is a human sdAb produced by a transgenic mouse or rat that expresses a human heavy chain segment. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794. In some embodiments, the sdAb is affinity matured.

[0276] In some embodiments, the naturally occurring V H H domain for a particular antigen or target is a camelid V HIt can be obtained from a (natural or immune) library of the H array. Such methods may or may not involve screening of such libraries using one or more screening techniques originally known, using the antigen or target, or at least one portion, fragment, epitope, or its epitope. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, for example, by techniques such as random mutagenesis and / or CDR shuffling described in WO00 / 43507, a (natural or immune) V H H library-derived V H H library, etc. (natural or immune) V H An improved synthetic or semi-synthetic library derived from the H library can be used.

[0277] In some embodiments, the sdAb is generated from a conventional four-chain antibody. For example, see EP 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 is / are cell surface molecules. The binding moiety (such as sdAb, etc.) can be selected to recognize an antigen that serves as a cell surface marker on target cells associated with a particular pathological condition. In some embodiments, the antigen (such as the first antigen and / or the second antigen, etc.) is a tumor antigen. In some embodiments, the multispecific CAR targets two or more tumor antigens. In some embodiments, the tumor antigen is associated with B cell malignancies. Tumors express several proteins that can function as target antigens for an immune response, specifically a 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 each contributing 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 induce an immune response, specifically a T cell-mediated immune response. The selection of the 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, alphafetoprotein (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-la, p53, prostain, 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 a malignant tumor. Malignant tumors express several proteins that can function as target antigens for an immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostate 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 cancer fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen unique to an individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B-cell lymphoma.

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

[0282] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-series antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant 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 antigens based on large proteins include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 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 (such as the first antigen and / or the second antigen, etc.) 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 The various binding moieties (such as sdAb) in the multispecific or multivalent CARs described herein can be fused to each other via a peptide linker. In some embodiments, the binding moieties (such as sdAb) are fused directly to each other without any peptide linker. The peptide linkers that link different binding moieties (such as sdAb) may be the same or different. Different domains of the CAR can also be fused to each other via a peptide linker.

[0285] Each peptide linker in the CAR can have the same or different lengths and / or sequences depending on the structural and / or functional characteristics of the sdAb and / or the 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 the CAR can affect properties including, but not limited to, the affinity, specificity, or binding strength to 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 hinder each other. For example, in the multivalent or multispecific CARs of the present application that include sdAbs directed against multimeric antigens, the length and flexibility of the peptide linker are preferably such that each sdAb in the multivalent CAR can bind to the antigenic determinants on each of the subunits of the multimer. In some embodiments, a short peptide linker can be placed between the transmembrane domain of the CAR and the intracellular signaling domain. In some embodiments, the peptide linker includes flexible residues (such as glycine and serine) such that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can 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 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, or any of the lengths therebelow of amino acids. In some embodiments, the length of the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids in length, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids, any of which.

[0287] The peptide linker can 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 can be used as a linker. See, e.g., 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) n , wherein 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 is an amino acid It comprises the sequences 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 CARs of the present application include a transmembrane domain that can be fused directly or indirectly to an extracellular antigen-binding domain. The transmembrane domain can be derived from either a natural or a synthetic source. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable within a cell membrane, preferably within a eukaryotic cell membrane. Transmembrane domains suitable for use in the CARs 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 within a cell membrane.

[0289] Transmembrane domains are classified based on their three-dimensional structure. For example, a transmembrane domain 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 cell's phospholipid bilayer. Additionally, or alternatively, transmembrane domains can be classified based on transmembrane domain topology, which includes the number of times the transmembrane domain crosses the membrane and the orientation of the protein. For example, a single-pass membrane protein crosses the cell membrane once, and a multi-pass membrane protein crosses 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 the topology of their termini and membrane-spanning segment(s) with respect to the inside and outside of the cell. Type I membrane proteins have a region spanning a single membrane and are oriented such that the N-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the C-terminus of the protein is on the cytoplasmic side. Type II membrane proteins also have a region spanning a single membrane but are oriented such 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 segments spanning multiple membranes and can be further subclassified based on the number of transmembrane segments and the positions of the N- and C-termini.

[0290] In some embodiments, the transmembrane domain of the CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains derived from multi-pass membrane proteins may also be suitable for use in the CARs described herein. Multi-pass membrane proteins can include composite (at least two, three, four, five, six, seven, or more) alpha helix or beta sheet structures. Preferably, the N-terminus and C-terminus of the multi-pass membrane protein are on opposite sides of the lipid bilayer. For example, 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 the transmembrane domain of the alpha chain, beta chain, 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 (KLRF1), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD1 8, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), 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 is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, 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 the transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO: 193. In some embodiments, the transmembrane domain of CD8α is encoded by the nucleic acid sequence of SEQ ID NO: 202.

[0294] The transmembrane domain for use in the CARs described herein may also include 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, such as 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 and are found, for example, in 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 include a transmembrane region and a cytoplasmic region located on the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may include three or more amino acids and, in some embodiments, helps to orient 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 includes positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain includes 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 the CAR comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises mainly 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 the Kyte and Doolittle hydropathy analysis.

[0297] Intracellular signaling domain The CARs of the present application include an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one of the normal effector functions of the 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 cytokine secretion. Thus, the term "cytoplasmic signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Usually, the entire cytoplasmic signaling domain can be used, but in many 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 a truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. Thus, the term cytoplasmic signaling domain is intended to include any truncated portion of the cytoplasmic signaling domain that is sufficient to transduce the 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. The "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 an immunoreceptor tyrosine-based activation motif or a signaling motif known as an ITAM. As used herein, "ITAM" is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. This motif can include two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid that results in the conserved motif YxxL / Ix(6-8)YxxL / I. The ITAM within a signaling molecule is required for intracellular signal transduction, which is at least partially mediated by phosphorylation of tyrosine residues within the ITAM after activation of the signaling molecule. The ITAM can also function as a docking site for other proteins involved in the signaling pathway. 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 that includes one or more mutations such as Q65K. In some embodiments, the primary intracellular signaling domain of the 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] Co-stimulatory signaling domain Many immune effector cells require co-stimulation in addition to stimulation by antigen-specific signals in order to promote cell proliferation, differentiation, and survival and to activate the effector functions of the cells. In some embodiments, the CAR includes at least one co-stimulatory signaling domain. As used herein, the term "co-stimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as an effector function. The co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein that transduces signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "co-stimulatory signaling domain" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand and thereby mediates a co-stimulatory response by immune cells, such as proliferation and survival, but is not limited thereto. As used herein, the term "co-stimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as an effector function. The co-stimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a co-stimulatory protein that transduces signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "co-stimulatory signaling domain" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand and thereby mediates a co-stimulatory response by immune cells, such as proliferation and survival, but is not limited thereto.

[0301] In some embodiments, the intracellular signaling domain comprises a single co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (such as about two, three, four, or any of more thereof) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same co-stimulatory signaling domain, for example, two copies of the co-stimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains from different co-stimulatory proteins such as any two or more of the co-stimulatory 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 co-stimulatory signaling domains. In some embodiments, the one or more co-stimulatory 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 the one or more co-stimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3ζ). Multiple co-stimulatory signaling domains can provide an additive or synergistic stimulatory effect.

[0302] Activation of a co-stimulatory 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 co-stimulatory signaling domain of any co-stimulatory molecule may be suitable for use in the CARs described herein. The type(s) of co-stimulatory signaling domain are selected based on factors such as the type of immune effector cell in which the effector molecule is expressed (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil), and the desired immune effector function (e.g., ADCC effect). Examples of co-stimulatory signaling domains for use in CARs are 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 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 TNF RII / 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 / The cytoplasmic signaling domains of co-stimulatory proteins can be, but are not limited to, SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150, and any other co-stimulatory molecules, such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha4 / CD49d, integrin alpha4beta1, integrin alpha4beta7 / 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 (LFA-1), and NKG2C.

[0303] In some embodiments, one or more co-stimulatory signaling domains are selected from the group consisting of ligands that specifically bind 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 the CAR of the present application includes a co-stimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain includes the cytoplasmic signaling domain of CD3ζ and the co-stimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain includes the co-stimulatory signaling domain of CD28 comprising the amino acid sequence of SEQ ID NO: 195. In some embodiments, the co-stimulatory 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 the CAR of the present application includes a co-stimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain includes the cytoplasmic signaling domain of CD3ζ and the co-stimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain includes the co-stimulatory signaling domain of CD137 comprising the amino acid sequence of SEQ ID NO: 196. In some embodiments, the co-stimulatory 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 the CAR of the present application includes the co-stimulatory signaling domain of CD28 and the co-stimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain includes the cytoplasmic signaling domain of CD3ζ, the co-stimulatory signaling domain of CD28, and the co-stimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain includes, from the N-terminus to the C-terminus, the co-stimulatory signaling domain of CD28, the co-stimulatory signaling domain of CD137, and the cytoplasmic signaling domain of CD3ζ. In some embodiments, the co-stimulatory signaling domain of CD28 comprising the amino acid sequence of SEQ ID NO: 195. In some embodiments, the co-stimulatory signaling domain of CD137 comprising the amino acid sequence of SEQ ID NO: 196.

[0307] So that the co-stimulatory signaling domain can regulate the immune response of immune cells, any variant of the co-stimulatory signaling domains described herein is also within the scope of the present disclosure. In some embodiments, the co-stimulatory signaling domain includes up to 10 amino acid residue modifications (e.g., 1, 2, 3, 4, 5, or 8) compared to the wild-type counterpart. Such co-stimulatory signaling domains that include one or more amino acid modifications may be referred to as variants. The amino acid residue variations of the co-stimulatory signaling domain result in an increase in signal transduction transfection and an increase in the stimulation of the immune response compared to the co-stimulatory signaling domain without the variation. can provide strength. Mutations in the amino acid residues of the co-stimulatory signaling domain can result in a decrease in signal transduction transduction and a reduction in the stimulation of the immune response compared to the co-stimulatory signaling domain without the mutation.

[0308] hinge region The CAR of the present application may include a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. The hinge domain is generally an amino acid segment found between two domains of a protein and may allow flexibility of the protein and movement of one or both of those domains relative to each other. 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 may contain any one of about 10 to 100 amino acids, such as 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 any one of 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 a hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art for containing a hinge domain is suitable for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a part of the hinge domain of a naturally occurring protein, imparting flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a fragment of the hinge domain of CD8α, containing, for example, at least 15 (e.g., 20, 25, 30, 35, or 40) contiguous amino acids of the hinge domain of CD8α. 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] The hinge domains of antibodies such as IgG, IgA, IgM, IgE, or IgD antibodies are also suitable for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, the hinge domain is the hinge domain that links the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge domain is that of an antibody comprising the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of the antibody and the CH2 and CH3 constant regions of the antibody. 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 of an IgG1 antibody and the CH2 and CH3 constant regions. In some embodiments, the hinge region comprises the hinge region of an IgG1 antibody and the CH3 constant region.

[0312] Non-naturally occurring peptides can also be used as the hinge domain of the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, e.g., a (GxS)n linker, wherein x and n are independently integers from 3 to 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 the polypeptide to a desired site within the cell. In some embodiments, the signal peptide targets an effector molecule to the secretory pathway of the cell and enables incorporation and retention of the effector molecule into the lipid bilayer. Signal peptides comprising signal sequences of naturally occurring proteins or synthetic non-naturally occurring signal sequences that are compatible for use in the CARs described herein will be apparent to those of skill 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 Also provided herein is a host cell (such as an immune effector cell) comprising any one of the CARs described herein.

[0315] Accordingly, in some embodiments, there are provided engineered immune effector cells (such as T cells) comprising 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, provided are engineered immune effector cells (such as T cells) comprising 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, wherein the first epitope and the second epitope are different; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the first anti-BCMA sdAb and / or the second anti-BCMA sdAb is camelid, chimeric, human, or humanized. In some embodiments, the first anti-BCMA and the second anti-BCMA are fused to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker is about 50 amino acids in length or less (such as any one of about 35, 25, 20, 15, 10, or 5 amino acids 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 the 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 co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of 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 the N-terminus to the C-terminus, a CD8α signal peptide, It includes an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory 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, there is provided an engineered immune effector cell (such as a T cell) comprising a BCMA CAR 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 is any of the following: (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 2, 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: 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 a CDR3 comprising the amino acid sequence of SEQ ID NO: 89, (14) a CDR1 comprising th...

Claims

1. A multivalent chimeric antigen receptor (CAR) comprising a polypeptide, (a) an extracellular antigen-binding domain comprising a first BCMA-binding portion and a second BCMA-binding portion, wherein the first BCMA-binding portion is a first anti-BCMA single-domain antibody (sdAb), and the second BCMA-binding portion is a second anti-BCMA sdAb; an extracellular antigen-binding domain, (b) a transmembrane domain, (c) an intracellular signaling domain, and the first anti-BCMA sdAb and the second anti-BCMA sdAb are each independently, (1) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 115, (2) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 117, (3) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 121, (4) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 124, and (5) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 125, and is an sdAb comprising any selected from the group consisting of, wherein the CDR1, CDR2, and CDR3 are defined by the Kabat numbering system, the multivalent chimeric antigen receptor (CAR).

2. The first anti-BCMA sdAb and the second anti-BCMA sdAb are each independently, (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR3 comprising the amino acid sequence of SEQ ID NO: 77, (2) 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, (3) 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, (4) 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, and (5) 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, The multivalent CAR according to claim 1, which is an sdAb comprising any one selected from the group consisting of.

3. The first anti-BCMA sdAb and the second anti-BCMA sdAb are each a V Ｈ H domain, and The first anti-BCMA sdAb and the second anti-BCMA sdAb each optionally and independently comprise any one of the amino acid sequences of SEQ ID NOs: 115, 117, 121, and 124-125, The multivalent CAR according to claim 1.

4. The first anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 defined in the anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 115, and The second anti-BCMA sdAb comprises CDR1, CDR2, and CDR3 defined in the anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 115, The multivalent CAR according to claim 1.

5. (i) The first anti-BCMA sdAb comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and CDR3 comprising the amino acid sequence of SEQ ID NO: 77, and (ii) The multivalent CAR according to claim 4, wherein the second anti-BCMA sdAb comprises 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. **Claim 6** The first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 115, and The multivalent CAR according to claim 4, wherein the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO:

115. **Claim 7** The first anti-BCMA sdAb comprises a CDR1, a CDR2, and a CDR3 defined in the anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 124, and The second anti-BCMA sdAb comprises a CDR1, a CDR2, and a CDR3 defined in the anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 125, The multivalent CAR according to claim 1. **Claim 8** (i) 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 (ii) The multivalent CAR according to claim 7, wherein the second 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. **Claim 9** The first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 124, and The multivalent CAR according to claim 7, wherein the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO:

125. **Claim 10** The first anti-BCMA sdAb comprises a CDR1, a CDR2, and a CDR3 defined in the anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 121, and the second anti-BCMA sdAb comprises an anti-BCMA comprising the amino acid sequence of SEQ ID NO: 125 and comprises CDR1, CDR2 and CDR3 defined in the sdAb, The multivalent CAR according to claim 1.

11. (i) 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 (ii) the second 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, the multivalent CAR according to claim 10.

12. the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 121, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 125, the multivalent CAR according to claim 10.

13. the first anti-BCMA sdAb is located at the N-terminus of the second anti-BCMA sdAb, or the first anti-BCMA sdAb is located at the C-terminus of the second anti-BCMA sdAb, the multivalent CAR according to any one of claims 1 to 12.

14. the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, the multivalent CAR according to any one of claims 1 to 13.

15. the transmembrane domain is derived from CD8α or CD28, the multivalent CAR according to claim 14.

16. the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, the multivalent CAR according to any one of claims 1 to 15.

17. The multivalent CAR according to claim 16, wherein the primary intracellular signaling domain is derived from CD3ζ.

18. The multivalent CAR according to any one of claims 1 to 15, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain.

19. The multivalent CAR according to claim 18, wherein the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, the ligand of B7-H3, CD83, and combinations thereof.

20. The multivalent CAR according to claim 19, wherein the co-stimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

21. The multivalent CAR according to any one of claims 1 to 20, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

22. The multivalent CAR according to claim 21, wherein the hinge domain is derived from CD8α.

23. The multivalent CAR according to any one of claims 1 to 22, further comprising a signal peptide located at the N-terminus of the polypeptide.

24. The multivalent CAR according to claim 23, wherein the signal peptide is derived from CD8α.

25. A multivalent chimeric antigen receptor (CAR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 298, 299, 306-315, and 317.

26. The following: (1) CDR1, CDR2, and CDR3 defined in an anti-BCMA sdAb comprising the amino acid sequence of SEQ ID NO: 115 (2) CDR1, CDR2, and CDR3 defined in the anti-BCMA sdAb containing the amino acid sequence of SEQ ID NO: 117, (3) CDR1, CDR2, and CDR3 defined in the anti-BCMA sdAb containing the amino acid sequence of SEQ ID NO: 121, (4) CDR1, CDR2, and CDR3 defined in the anti-BCMA sdAb containing the amino acid sequence of SEQ ID NO: 124, and (5) CDR1, CDR2, and CDR3 defined in the anti-BCMA sdAb containing the amino acid sequence of SEQ ID NO: 125, comprising any one of the following: An anti-BCMA single-domain antibody (sdAb) wherein CDR1, CDR2, and CDR3 are defined by the Kabat numbering system.

27. The following: (1) CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 39, and CDR3 containing the amino acid sequence of SEQ ID NO: 77, (2) CDR1 containing the amino acid sequence of SEQ ID NO: 3, CDR2 containing the amino acid sequence of SEQ ID NO: 41, and CDR3 containing the amino acid sequence of SEQ ID NO: 79, (3) CDR1 containing the amino acid sequence of SEQ ID NO: 7, CDR2 containing the amino acid sequence of SEQ ID NO: 45, and CDR3 containing the amino acid sequence of SEQ ID NO: 83, (4) CDR1 containing the amino acid sequence of SEQ ID NO: 10, CDR2 containing the amino acid sequence of SEQ ID NO: 48, and CDR3 containing the amino acid sequence of SEQ ID NO: 86, and (5) CDR1 containing the amino acid sequence of SEQ ID NO: 11, CDR2 containing the amino acid sequence of SEQ ID NO: 49, and CDR3 containing the amino acid sequence of SEQ ID NO: 87, The anti-BCMA sdAb according to claim 26, comprising any one of the following.

28. The anti-BCMA sdAb according to claim 26, wherein the anti-BCMA sdAb contains any one of the amino acid sequences of SEQ ID NOs: 115, 117, 121, and 124-125.

29. The anti-BCMA sdAb is V Ｈ H domain, the anti-BCMA sdAb according to claim 26. sdAb.

30. A chimeric antigen receptor (CAR) comprising a polypeptide, comprising: (a) an extracellular antigen-binding domain comprising at least one anti-BCMA sdAb according to any one of claims 26 to 29; (b) a transmembrane domain; (c) an intracellular signaling domain, wherein the chimeric antigen receptor (CAR) comprises the transmembrane domain.

31. The transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD8 6, CD152, and PD1, the CAR according to claim 30.

32. The transmembrane domain is derived from CD8α or CD28, the CAR according to claim 31.

33. The intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, the CAR according to any one of claims 30 to 32.

34. The primary intracellular signaling domain is derived from CD3ζ, the CAR according to claim 33.

35. The intracellular signaling domain comprises a co-stimulatory signaling domain, the CAR according to any one of claims 30 to 32.

36. The co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of CD83, and combinations thereof, the CAR according to claim 35.

37. The chimeric antigen receptor (CAR) according to claim 36, wherein the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137.

38. The CAR according to any one of claims 30 to 37, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

39. The CAR according to claim 38, wherein the hinge domain is derived from CD8α.

40. The CAR according to any one of claims 30 to 39, further comprising a signal peptide located at the N-terminus of the polypeptide.

41. The CAR according to claim 40, wherein the signal peptide is derived from CD8α.

42. A chimeric antigen receptor (CAR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 to 219 and 221 to 229.

43. An isolated nucleic acid comprising a nucleic acid sequence encoding a multivalent CAR according to any one of claims 1 to 25, an anti-BCMA sdAb according to any one of claims 26 to 29, or a CAR according to any one of claims 30 to 42.

44. A vector comprising the isolated nucleic acid according to claim 43.

45. An engineered immune effector cell comprising a multivalent CAR according to any one of claims 1 to 25 or a CAR according to any one of claims 30 to 42.

46. The engineered immune effector cell according to claim 45, wherein the immune effector cell is a T cell.

47. A pharmaceutical composition comprising the engineered immune effector cell according to claim 45 or 46 and a pharmaceutically acceptable carrier.

48. Use of an engineered immune effector cell according to claim 45 or 46, or an anti-BCMA sdAb according to any one of claims 26 to 29, in the manufacture of a medicament for treating cancer in an individual, wherein the cancer is a cancer comprising cells expressing BCMA. **Claim 49** Use according to claim 48, wherein the cancer is multiple myeloma. **Claim 50** Use according to claim 49, wherein the cancer is refractory or relapsed multiple myeloma.

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