Chimeric antigen receptor targeting BCMA and method of use thereof

Abstract

This application relates to an anti-BCMA single-domain antibody (sdAb), one or more anti-BCMA sdAbs (V H The objective is to provide chimeric antigen receptors (CARs) containing H fragments, manipulated immune effector cells, and methods for using them in cancer immunotherapy. [Solution] Anti-BCMA single-domain antibody (sdAb), one or more anti-BCMA sdAbs (V H We identified chimeric antigen receptors (CARs) containing H fragments, engineered immune effector cells, and methods for their use in cancer immunotherapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to International Patent Application PCT / CN2016 / 094408, filed on 10 August 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] Submission of sequence listings in ASCII text files. The following submission in ASCII text file is incorporated herein by reference in its entirety: a computer-readable format (CRF) sequence listing (filename: 761422000640SEQLISTING.txt, date: August 10, 2017, size: 520KB).

[0003] This invention relates to a single-domain antibody targeting BCMA, a chimeric antigen receptor, engineered immunoeffector cells, and methods for using the same. [Background technology]

[0004] With advancements in 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, chimeric antigen receptors (CARs) comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain may contain a single-stranded variable fragment (scFv) that targets a specific tumor antigen. CARs can be expressed on the surface of T cells using gene transfection techniques. Upon binding to a target tumor antigen, CARs can activate T cells to initiate a specific antitumor response in an antigen-dependent manner, without being limited by the availability of a major histocompatibility complex (MHC) specific to the target tumor antigen.

[0005] Single-domain antibodies (sdAbs) differ from conventional four-chain antibodies in that they possess a single monomeric antibody variable domain. For example, camelids and sharks produce sdAbs, which have been named heavy-chain-only antibodies (HcAbs), and these naturally lack a light chain. The antigen-binding fragment in each arm of a camelid heavy-chain-only antibody is a single heavy-chain variable domain (V H It possesses H), which allows it to have high affinity for the antigen without the aid of a light chain. Camelidae V H H is known as the smallest functional antigen-binding fragment with a molecular weight of approximately 15 kD.

[0006] Multiple myeloma (MM) is an incurable, invasive plasma malignancy that is classified as B-cell neoplasia, where cells proliferate uncontrollably within the bone marrow, interfering with the normal metabolic production of blood cells and causing painful bone lesions (Garfall, A et al., Discovery Med. 2014, 17, 37). Multiple myeloma can clinically present with hypercalcemia, renal failure, anemia, bone lesions, bacterial infections, hyperviscosity, and amyloidosis (Robert Z. Orlowski, Cancer Cell. 2013, 24(3)). Studies and statistics show that nearly 86,000 people are diagnosed with myeloma each year, while approximately 63,000 die each year from disease-related complications (Becker, 2011). Due to the aging population, the number of myeloma cases is projected 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 radiotherapy, stem cell transplantation or bone marrow transplantation, targeted therapy, or biological therapy (George, 2014). Antibody-based cellular immunotherapy has demonstrated substantial clinical benefit in patients with hematological malignancies, specifically B-cell non-Hodgkin lymphoma. Current therapies for multiple myeloma often result in remission, but almost all patients eventually relapse. There is a need for effective immunotherapeutic drugs to treat multiple myeloma.

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

[0008] Overall, the objective response rate was 100%, with 33 patients (94%) achieving clear clinical remission of myeloma (complete response, very good partial response, or partial response) within two months of receiving CAR T cells. Following this group for more than four months, in terms of efficacy, 14 of the 19 patients achieved stringent complete response, one achieved partial response, and four achieved very good partial remission.

[0009] The superior efficacy and safety profile obtained from the LCAR-B38M clinical trial is significantly better than several other BCMA CAR-T trials reported simultaneously at ASCO, which were widely recognized as a "revolutionary leap 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] All publications, patents, patent applications, and disclosures of published patent applications referenced herein are incorporated herein by reference in their entirety. [Overview of the project]

[0011] This application relates to an anti-BCMA single-domain antibody (sdAb), one or more anti-BCMA sdAbs (V HThis invention provides chimeric antigen receptors (CARs) containing H fragments, engineered immune effector cells, and methods for using them in cancer immunotherapy.

[0012] One aspect of this application provides an anti-BCMA sdAb comprising any one of the CDR regions of SEQ ID NOs: 115 to 152. In some embodiments, the anti-BCMA sdAb comprises: (1) 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) comprising the amino acid sequence of SEQ ID NO: 4 (5) CDR1 containing the amino acid sequence of SEQ ID NO: 42, and CDR3 containing the amino acid sequence of SEQ ID NO: 80, (6) CDR1 containing the amino acid sequence of SEQ ID NO: 5, CDR2 containing the amino acid sequence of SEQ ID NO: 43, and CDR3 containing the amino acid sequence of SEQ ID NO: 81, (7) CDR1 containing the amino acid sequence of SEQ ID NO: 6, CDR2 containing the amino acid sequence of SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 82, (8) CDR1 containing the amino acid sequence of SEQ ID NO: 7, and CDR2 containing the amino acid sequence of SEQ ID NO: 45, and (8) CDR3 containing the amino acid sequence of SEQ ID NO: 83, (9) CDR1 containing the amino acid sequence of SEQ ID NO: 8, CDR2 containing the amino acid sequence of SEQ ID NO: 46, and CDR3 containing the amino acid sequence of SEQ ID NO: 84, (10) CDR1 containing the amino acid sequence of SEQ ID NO: 9, CDR2 containing the amino acid sequence of SEQ ID NO: 47, and CDR3 containing the amino acid sequence of SEQ ID NO: 85, (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 C containing the amino acid sequence of SEQ ID NO: 93 DR3, (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, and the amino acid sequence of SEQ ID NO: 59 CDR2 containing the sequence, 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) SEQ ID NO: CDR1 containing the amino acid sequence of 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, and CDR2 containing the amino acid sequence of SEQ ID NO: 66,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 C containing the amino acid sequence of SEQ ID NO: 109 DR3, (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 V containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. H Includes the H domain.

[0013] In some embodiments, antigen-binding proteins are provided that contain either an anti-BCMA heavy chain-only antibody (HCAB) or one of the anti-BCMA sdAbs described above. Also provided are BCMA epitopes that specifically bind to an anti-BCMA antibody (such as an anti-BCMA sdAb) that competes with one of the anti-BCMA sdAbs described above.

[0014] In some embodiments, following 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 V H This is an H fragment.

[0015] One aspect of this application provides a polypeptide-based BCMA chimeric antigen receptor comprising (a) an extracellular antigen-binding domain containing an anti-BCMA sdAb (such as any one of the anti-BCMA sdAbs listed 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 polyvalent (such as bivalent or trivalent). In some embodiments, the CAR is multispecific (such as bispecific). In some embodiments, the extracellular antigen-binding domain contains at least two anti-BCMA sdAbs (such as any one or more of the anti-BCMA sdAbs listed above).

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

[0017] One aspect of this application provides a polyvalent (bivalent or trivalent, etc.) chimeric antigen receptor comprising a polypeptide, (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 and second anti-BCMA sdAb specifically bind to the same epitope on BCMA. In some embodiments, the first and second anti-BCMA sdAb specifically bind to different epitopes on BCMA. In some embodiments, the first and / or second anti-BCMA sdAb specifically bind to epitopes on BCMA derived from amino acid sequences selected from SEQ ID NOs. 388-394. In some embodiments, the first anti-BCMA sdAb specifically binds to epitopes derived from SEQ ID NOs. 389 and / or 390. In some embodiments, the second anti-BCMA sdAb specifically binds to epitopes derived from SEQ ID NOs. 391 and / or 392.

[0018] In some embodiments, following any one of the polyvalent 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 has an amino acid length of about 50 or fewer (any number such as about 35, 25, 20, 15, 10, or 5 or fewer). In some embodiments, the peptide linker contains an amino acid sequence selected from SEQ ID NOs. 208-215.

[0019] In some embodiments, following any one of the CARs (including polyvalent 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 contains the amino acid sequence of SEQ ID NO: 193 or 194.

[0020] In some embodiments, following any one of the CARs (including polyvalent CARs) described above, 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 primary intracellular signaling domain includes the amino acid sequence of SEQ ID NO: 197 or 198.

[0021] In some embodiments, following any one of the CARs (including polyvalent CARs) described above, 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the co-stimulatory signaling domain includes the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137. In some embodiments, the co-stimulatory signaling domain includes the amino acid sequence of SEQ ID NO: 195 and / or SEQ ID NO: 196.

[0022] In some embodiments, following any one of the CARs (including polyvalent 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 includes the amino acid sequence of SEQ ID NO: 192.

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

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

[0025] One aspect of this 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 this application provides an isolated nucleic acid comprising a nucleic acid sequence encoding either anti-BCMA sdAb or CAR (including polyvalent CAR) as 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 this application provides a vector comprising any one of the isolated nucleic acids described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a nonviral vector.

[0028] One aspect of this application provides engineered immune effector cells comprising any one of the CARs (including polyvalent 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 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 immune effector cells are T cells.

[0029] One aspect of this application provides a pharmaceutical composition comprising any one of the engineered immune effector cells described above and a pharmaceutically acceptable carrier. A method for 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 liquid cancer. In some embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia. In some embodiments, the cancer is solid cancer such as glioblastoma. In some embodiments, the cancer is refractory or recurrent multiple myeloma.

[0030] One aspect of this application relates to any one of the anti-BCMA sdAb described above, and a drug The present invention provides a pharmaceutical composition comprising a scientifically acceptable carrier. In some embodiments, a method for treating a disease (such as cancer) in an individual is further provided, which includes administering an effective amount of the pharmaceutical composition to the individual.

[0031] Methods of use, kits, and products are also provided, each containing one of the following: anti-BCMA sdAb, CAR (including polyvalent CAR), engineered immune effector cells, isolated nucleic acids, or vectors. [Brief explanation of the drawing]

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

[0033] This application provides anti-BCMA single-domain antibodies (sdAbs) and chimeric antigen receptors (CARs) comprising an extracellular antigen-binding domain containing one or more BCMA-binding moieties (e.g., anti-BCMA sdAbs). Also provided are polyvalent CARs comprising at least two binding moieties (e.g., sdAbs) that specifically bind to a single antigen. In some embodiments, this application provides polyvalent (e.g., bivalent or trivalent) CARs comprising at least two anti-BCMA sdAbs. In some embodiments, the at least two anti-BCMA sdAbs are different anti-BCMA sdAbs that specifically bind to different epitopes on BCMA. Anti-BCMA sdAbs, CARs, and engineered immune effector cells expressing the CARs described in this application are useful agents for cancer treatment.

[0034] In particular, this application demonstrates the superior efficacy of a bivalent, bivalent epitope CAR (e.g., LCAR-B38M) containing two anti-BCMA sdAbs targeting different BCMA epitopes in treating multiple myeloma in human patients. In an interim analysis of a Phase I / II clinical trial, patients with relapsed or refractory multiple myeloma responded 100% to LCAR-B38M CAR-T therapy. 94% of patients achieved clear clinical remission of myeloma within two months of receiving CAR-T therapy. Patients who achieved stringent complete response (sCR) maintained minimal residual disease-free status more than one year after receiving CAR-T therapy. Furthermore, LCAR-B38M CAR-T therapy was well-tolerated by patients, as most patients experienced only mild and manageable cytokine release syndrome, a common side effect of CAR-T cell-based therapies. Patients did not experience any neurological side effects. Relatively, pilot clinical studies of monovalent CARs containing a single anti-BCMA sdAb showed lower objective response and complete remission rates, as well as higher relapse rates among treated patients. Prior to this application, all BCMA CARs under clinical research had only one BCMA binding moiety in their extracellular antigen-binding domain. The improved clinical efficacy and safety of the multivalent BCMA CARs in this application are unexpected.

[0035] Accordingly, one aspect of this application provides a polyvalent CAR comprising a polypeptide including (a) an extracellular antigen-binding domain containing 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 embodiment, a polyvalent CAR is provided, comprising a polypeptide including (a) an extracellular antigen-binding domain including 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] Also described herein are engineered immune effector cells (such as T cells) containing CARs, and pharmaceutical compositions, kits, products, and methods for treating cancer using engineered immune effector cells or sdAbs.

[0039] I. Definition The term "antibody" includes monoclonal antibodies (including full-length four-chain antibodies with 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 herein as synonymous with "antibody." Antibodies as used herein include single-domain antibodies such as heavy-chain-only antibodies.

[0040] A basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called a J chain, containing 10 antigen-binding sites. IgA antibodies, on the other hand, are composed of 2 to 5 basic four-chain units, which can polymerize to form multivalent aggregates with the J chain. In the case of IgG, a four-chain unit generally has a value of approximately 150,000 daltons. Each L chain is linked to the H chain by a single 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. Both the H and L chains also have regularly spaced interchain disulfide bridges. Each H chain has a variable domain (V) at its N-terminus. H ) has, and subsequently, each of the α and γ chains has three constant domains (C H) and for the μ and ε isotypes there are four C H domains. Each L chain has a variable domain (V ) at the N-terminus and then a constant domain at the opposite end. V L is aligned with V L , and C H is aligned with the first constant domain of the heavy chain (C L 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 H 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. Depending on the amino acid sequence of their heavy chain constant domains (C H ), immunoglobulins can be assigned to different classes or isotypes. 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. H

[0041] The term "heavy chain only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chains 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 can bind to an antigen independently, without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy-chain variable domains are referred to herein as "V H It is called "H". Several V H H may also be known as a nanobody. Camelidae sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., 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)). 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-FR4 refer to framework regions 1-4, and CDR1-CDR3 refer to complementarity determination regions 1-3.

[0043] An “isolated” antibody is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from association with all other components from its production environment. Contaminations from its production environment, such as those resulting from recombinant transfected cells, are materials that would typically interfere with the study, diagnostic, or therapeutic use of the antibody, and these may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide would be purified to (1) more than 95% by weight of the antibody, and in some embodiments more than 99% by weight, as determined, for example, by the Lowry method; (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) to a degree that homogeneity is obtained by SDS-PAGE under non-reducible or reducing conditions using Coomassie blue, or preferably silver staining. Since the isolated antibody is free from at least one component of the antibody’s natural environment, it contains the antibody of insight in recombinant cells. However, usually isolated polypeptides The drug 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 chain or light chain of the antibody. The variable domains of the heavy chain and light chain are respectively called "V H " and "V L These domains can be referred to as "V". 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. Heavy chain-only antibodies from camelid species have a single heavy chain variable region, which is "V H It is called "H". Therefore, V H H is V H It is a special type.

[0045] The term "variable" refers to the fact that a particular segment of the variable domain has a significantly different sequence depending on the antibody. The V domain mediates antigen binding and defines the specificity of a particular antibody to that particular antigen. However, variability is not evenly distributed throughout the variable domain. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). Each of the natural heavy and light chain variable domains contains four FR regions that largely adopt a beta-sheet configuration linked by three HVRs that connect beta-sheet structures and, in some cases, form loops that form part of the beta-sheet structure. The HVRs of each chain are held together in very close proximity by the FR region and, together with the HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0046] When used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population; that is, the individual antibodies in that population are identical except for any naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in trace 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 an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture and are free from contamination with other immunoglobulins. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous antibody population and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with this application are, for example, those produced 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, NY, 1981)), Recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567), Phage display technology (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 See 101(34):12467-12472(2004); and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004), and techniques for producing human antibodies or human-like antibodies in animals possessing a human immunoglobulin locus or part or all of a human immunoglobulin gene encoding a human immunoglobulin sequence (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 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.It can be produced by various techniques, including (see 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 has not been conjugated with a cytotoxic moiety or radiolabeling.

[0048] The terms "full-length antibody," "intact antibody," or "whole antibody" are used synonymously to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, this includes antibodies that have a heavy chain and light chain containing the full-length 4-chain antibody Fc region. A full-length heavy-chain-only antibody is one in which the heavy chain (V) is located. H It includes H (etc.) and Fc regions. The constant domain may be the constant domain of the natural sequence (e.g., the constant domain of the human natural sequence) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0049] An "antibody fragment" is a portion of an intact antibody, preferably comprising the antigen-binding region and / or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see 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 H Examples include multispecific antibodies formed from antibody fragments (H, etc.). Papain digestion of the antibody yielded two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragment, denoted to reflect its ability to readily crystallize. The Fab fragment consists of the entire L chain, as well as the variable region domain (V) of the H chain. H ), as well as the first constant domain of one heavy chain (C H1) consists of each Fab fragment, which is monovalent with respect to antigen binding, i.e., has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which is roughly equivalent to two Fab fragments with different antigen-binding activities disulfide-linked together, and is still capable of crosslinking to an antigen. The Fab' fragment contains one or more cysteine ​​from the antibody hinge region, C H It differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of one domain. Fab'-SH is the heretical notation for Fab' in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair with a Fab' fragment that has a hinged cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0050] The Fc fragment contains the carboxyl ends of both H chains held together by a disulfide. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by an Fc receptor (FcR) found in certain cell types.

[0051] "Fv" is the smallest antibody fragment containing both a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly bound noncovalently. The folding of these two domains creates six hypervariable loops (three from the H chain and three from the 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 Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, although with lower affinity than the full binding site.

[0052] "Single-stranded Fv," also abbreviated as "sFv" or "scFv," is a single polypeptide chain formed by linking together V molecules. H and V LIt is an antibody fragment containing an antibody domain. Preferably, the sFv polypeptide is V H and V L The sFv further contains polypeptide linkers between its domains, enabling it to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg See and Moore eds., Springer-Verlag, New York, pp. 269–315 (1994).

[0053] The “functional fragments” of antibodies described herein include a portion of an intact antibody, which generally includes the antigen-binding region or variable region of an intact antibody, or the Fc region of an antibody that retains or modifies 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 the process of achieving V-domain pairing between chains, rather than within a chain, thereby obtaining a bivalent fragment, i.e., a fragment with two antigen-binding sites. H and V L This refers to a small antibody fragment prepared by constructing an sFv fragment (see previous paragraph) using a short linker (approximately 5-10 residues) between domains. A bispecificity diabody is a V of two antibodies. H and V L It is a heterodimer of two “crossed” sFv fragments whose domains are located on polypeptide chains with different domains. For more information on the diabody, see, for example, European Patent No. 404,097, International Publication No. WO93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0055] The monoclonal antibodies used herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is derived from a particular species or is identical or identical to a corresponding sequence in an antibody belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or identical to a corresponding sequence in an antibody belonging to a different species or another antibody class or subclass, as well as fragments of such antibodies, 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)). The chimeric antibodies used herein include PRIMATTZFD® antibodies, in which the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing macaque monkeys with the target antigen. As used herein, “humanized antibodies” are used as a subset of “chimeric antibodies.”

[0056] The "humanized" form of a non-human (e.g., camelid) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In some embodiments, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVR (defined below) are replaced with residues from the HVR of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capabilities. In some cases, framework ("FR") residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications may be made to further improve the antibody's performance, such as binding affinity. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, where all or substantially all of the hypervariable loops correspond to non-human immunoglobulin sequences, and all or substantially all of the FR regions correspond to human immunoglobulin sequences, although the FR regions may contain one or more individual FR residue substitutions that improve antibody performance such as binding affinity, isomerization, and immunogenicity. The number of these amino acid substitutions in the FR is typically six or less in the H chain and three or less in the L chain. Humanized antibodies also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of human immunoglobulin. For further details, see, for example, 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, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Patent Nos. 6,982,321 and 7,087,409.

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

[0058] As used herein, the terms “hypervariable region,” “HVR,” or “HV” refer to regions of antibody variable domains whose sequences are hypervariable and / or form structurally defined loops. Generally, sdAbs include three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 exhibits the highest variability of the three HVRs and is thought to play a unique role in conferring superior specificity to antibodies. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0059] The terms "complementarity-determining region" or "CDR" are used to refer to hypervariable regions as defined by the Kabat formula. 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 incorporated herein. The Kabat complementarity-determining region (CDR) is based on sequence variability and is 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, on the other hand, indicates the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on the analysis of available complex crystal structures. The residues of each of these HVRs are listed in Table 1 below. [Table 1]

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

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

[0063] The expressions “Kabat-like variable domain residue numbering” or “Kabat-like amino acid position numbering,” and their variations, refer to the numbering system used for heavy-chain or light-chain variable domains in antibody organization by Kabat et al. (above). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions of FR or HVR in the variable domain. For example, a heavy-chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and a residue inserted after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). Kabat numbering of residues can be determined for a given antibody by matching the homologous region between the antibody sequence and the “standard” Kabat-numbered sequence.

[0064] Unless otherwise specified herein, the numbering of residues within immunoglobulin heavy chains is that of the EU index, as presented in Kabat et al. above. "EU index, as presented in Kabat" refers to the residue numbering of human IgG1 EU antibodies.

[0065] A “framework” or “FR” residue is a variable domain residue other than an HVR residue as defined herein.

[0066] The "Human Consensus Framework" or "Acceptor Human Framework" is based on human immunoglobulin V L or V H This framework represents the most commonly occurring amino acid residues in the selection of framework sequences. Generally, it is used in human immunoglobulin V. L or V H Sequence selection is from subgroups of variable domain sequences. Generally, sequence subgroups are defined as follows: Kabat et al., Sequences of Proteins of Immunological Interest, 5 thThese are subgroups like those found in Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). For example, V L This includes those relating to the above, and the subgroups may be subgroups Kappa I, Kappa II, Kappa III, or Kappa IV, as described in Kabat et al. Furthermore, for VH, the subgroups may be subgroup I, subgroup II, or subgroup III, as described in Kabat et al. Alternatively, the human consensus framework may derive from the above, for example, when human framework residues are selected based on their homology to the donor framework by aligning specific residues, e.g., donor framework sequences, with a set of various human framework sequences. The human immunoglobulin framework or the acceptor human framework "derived from" the human consensus framework may contain the same amino acid sequence, or it may contain existing amino acid sequence changes. 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 specified location in the Fc region refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to the specified residue means an insertion within one or two residues. The insertion may be at the N-terminus or C-terminus of the specified residue. The preferred amino acid modification in this specification is substitution.

[0068] A "affinity-matured" antibody has one or more changes in one or more HVRs, and these changes result in improved antibody affinity to the antigen compared to a parent antibody that does not have those changes. In some embodiments, affinity-matured antibodies are used to improve the affinity of the target antigen. It has nanomolar or even picomolar affinity. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) H and V L Affinity maturation by domain shuffling is described. Random mutagenesis of HVR 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 bind,” “specifically recognize,” or “specific to” refer to measurable and reproducible interactions, such as binding, between a target and an antigen-binding protein (CAR or sdAb, etc.), which confirm the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antigen-binding protein (CAR or sdAb, etc.) that specifically binds to a target (which may be an epitope) is an antigen-binding protein (CAR or sdAb, etc.) that binds to this target more readily and / or for a longer duration with higher affinity, binding strength, than it would to other targets. In some embodiments, the extent to which an antigen-binding protein (CAR or sdAb, etc.) binds to an unrelated target is less than about 10% of that of the antigen-binding protein (CAR or sdAb, etc.), as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antigen-binding protein (CAR or sdAb, etc.) that specifically binds to the 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 (CAR or sdAb, etc.) specifically binds to an epitope on the protein that is conserved between proteins of different species. In some embodiments, specific binding may include, but is not required, exclusive binding.

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

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

[0072] The "effector function" of an antibody refers to the biological activity attributed to the antibody's Fc region (either the Fc region of the natural sequence or the Fc region of a variant amino acid sequence), and is diverse depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and cell surface receptors (for example...). Examples include downregulation of B cell receptors and B cell activation. "Reduced or minimized" antibody effector function indicates that it is reduced by at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from wild-type or unmodified antibodies. Determining antibody effector function is readily determinable and measurable by those skilled in the art. In preferred embodiments, antibody effector functions of complement binding, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity are affected. In some embodiments, effector function is eliminated through mutations in the constant region that exclude glycosylation, e.g., "effectorless mutations". In one embodiment, the effectorless mutation is C HThese are N297A or DANA mutations (D265A+N297A) in two regions. 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 may be reduced or eliminated through production techniques such as expression in non-glycosylating host cells (e.g., E. coli) or in host cells that result 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 immunoglobulin (Ig) bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them using cytotoxicity. Antibodies are necessary to "arm" cytotoxic cells and kill target cells through this mechanism. Primary cells that mediate ADCC, such as NK cells, express only FcγRIII, while 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 target molecule, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a target molecule may be evaluated in vivo in animal models, such as those disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0074] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, and includes both naturally occurring and mutant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or from 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 recombination of the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition may include an antibody population from which all K447 residues have been removed, an antibody population without any removed K447 residues, and an antibody population having a mixture of antibodies with and without K447 residues. Suitable naturally occurring 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 strength of the combined 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 herein, "binding affinity" refers to the members of the binding pair. For example, it refers to the intrinsic binding affinity, which reflects the 1:1 interaction between an antibody and an antigen (or a CAR and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them 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 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 peptide, polypeptide, or antibody sequences, "amino acid sequence identity percentage (%)" and "homology" are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after sequence alignment and the introduction of gaps as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the entire 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 graph one or more antigen specificities in immune effector cells such as T cells. Some CARs are also known as “artificial T cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” In some embodiments, a CAR comprises one or more antigens (such as tumor antigens) of T cells and / or other receptors, a transmembrane domain, and an extracellular antigen-binding domain specific to an intracellular signaling domain. “CAR-T” refers to a T cell expressing a CAR. “BCMA CAR” refers to a CAR having an extracellular antigen-binding domain specific to BCMA. “Dual epitope CAR” refers to a CAR having extracellular binding domains specific to two different epitopes on BCMA.

[0079] The “isolated” nucleic acid molecules encoding CARs or sdAbs described herein are nucleic acid molecules identified and separated from at least one contaminating nucleic acid molecule that would normally associate with the environment in which they were produced. Preferably, the isolated nucleic acid does not associate with any components associated with the production environment. The isolated nucleic acid molecules encoding polypeptides and antibodies herein are in forms other than those found in nature or in settings. Thus, the isolated nucleic acid molecules are distinct from the nucleic acids encoding polypeptides and antibodies herein that are naturally present in cells.

[0080] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a manipulably linked coding sequence in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0081] Nucleic acids are "operably linked" when they are placed in a functional relationship with another nucleic acid sequence. For example, if a pre-sequence or secretion leader DNA is expressed as a preprotein involved in polypeptide secretion, it is operably ligated to the polypeptide DNA; if a promoter or enhancer affects the transcription of the coding sequence, it is operably ligated to that sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably ligated to the coding sequence. Generally, "operably ligated" means that the ligated DNA sequence is contiguous, and in the case of a secretion leader, it is contiguous and in the leading phase. However, enhancers do not have to be contiguous. Ligation is achieved by ligation at a convenient restriction site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to 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 bound. 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 can induce the expression of the nucleic acid to which they are manipulably linked. Such vectors are referred to herein as “expression vectors.”

[0083] As used herein, the term “self-derived” is intended to refer to any material that originates from the same individual from which it is later reintroduced.

[0084] "Same species" refers to grafts derived from different individuals of the same species.

[0085] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. This includes primary target cells and their offspring.

[0086] As used herein, the terms “cell,” “cell line,” and “cell culture” are used synonymously, and all such designations include offspring. Therefore, “transfectant” and “transfected cell” include the primary target cells and any culture derived therefrom, regardless of the number of transfections. It is also understood that all offspring may not be strictly identical in terms of DNA content due to intentional or accidental mutations. This includes mutant offspring having the same function or biological activity as those screened for the initially transformed cells.

[0087] The terms “host cell,” “host cell line,” and “host cell culture” are used synonymously and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Examples of host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. The offspring may contain mutations, although they may not be entirely identical to the parent cells in terms of nucleic acid content. Mutant offspring having the same function or biological activity as those screened or selected from the initially transformed cells are included herein.

[0088] As used herein, “treatment” or “to treat” means an approach to obtain beneficial or desired outcomes, including clinical outcomes. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by the disease, reduction of the severity of the disease, stabilization of the disease (e.g., prevention or delay of disease exacerbation), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of the condition, provision of remission (partial or total) of the disease, reduction of the dose of one or more other drugs required to treat the disease, delay of disease progression, improvement of quality of life, and / or extension of survival. The methods of this application aim to achieve one or more of these therapeutic embodiments.

[0089] As used herein, “individual” or “subject” refers to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0090] As used herein, the term “effective dose” refers to an amount of a drug, e.g., sdAb, engineered immune effector cells, or a pharmaceutically effective composition thereof, sufficient to treat a particular disorder, condition, or disease, for example, to improve, alleviate, reduce, and / or delay one or more of its symptoms. With respect to cancer, an effective dose includes an amount sufficient to cause tumor reduction and / or reduce the rate of tumor growth (e.g., suppress tumor growth) or to inhibit or delay other undesirable cell proliferation. In some embodiments, an effective dose is sufficient to delay the onset of the disease. In some embodiments, an effective dose is sufficient to prevent or delay recurrence. An effective dose may be administered in one or more doses. An effective amount of drug or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) block, delay, to some extent delay, and preferably halt, cancer cell infiltration into peripheral organs, (iv) block tumor metastasis (i.e., to some extent delay, and preferably halt), (v) block tumor growth, (vi) prevent or delay tumor development and / or recurrence, and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer.

[0091] "Adjuvant situation" refers to a clinical situation in which an individual has a history of cancer and is responding to therapies, including but not limited to surgery (e.g., resection), radiation therapy, and chemotherapy. However, due to the individual's history of cancer, these individuals are considered to be at risk of developing the disease. Treatment or administration in an "adjuvant situation" refers to the subsequent mode of treatment. The degree of risk (e.g., whether an individual in an adjuvant situation is considered "high-risk" or "low-risk") depends on several factors, most commonly the severity of the disease at the time of initial treatment.

[0092] "Neo-adjuvant situation" refers to a clinical situation in which this method is used before primary / causal therapy.

[0093] As used herein, “delaying” the onset of cancer means withholding, interfering with, delaying, postponing, stabilizing, and / or postponing the onset of the disease. This delay may vary in duration depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, sufficient or significant delay may encompass prevention in that the individual does not, in effect, develop the disease. A method for “delaying” the onset of cancer is a method that reduces the likelihood of developing the disease and / or the severity of the disease within a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. Cancer onset may be detectable using standard methods including, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasonography, coagulation tests, angiography, or biopsy. Onset also refers to cancer progression, which may be undetectable in its early stages, and includes development, recurrence, and onset.

[0094] The term "pharmaceutical preparation" refers to a preparation that is in a form that enables the biological activity of the active ingredient and does not contain any further components that are unacceptably toxic to the subject to which the preparation is administered. Such preparations are sterile. A "sterile" preparation is either sterile or free from all viable microorganisms and their spores.

[0095] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations used. In many cases, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers Examples include 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 “diluent” referred to herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparations of liquid formulations, such as formulations to be reconstituted after lyophilization. Examples of diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextrose solutions. In an alternative embodiment, the diluent may include aqueous solutions of salts and / or buffer solutions.

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

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

[0099] A “reconstituted” formulation is prepared by dissolving a lyophilized protein or antibody formulation in a diluent so that the protein is evenly dispersed. Reconstituted formulations are suitable for administration to patients treated with the target protein (e.g., subcutaneous administration) and, in some embodiments of this application, may be suitable for parenteral or intravenous administration.

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

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

[0102] References to values ​​or parameters “about” in this specification include (and describe) variations relating to the value or parameter itself. For example, a statement referring to “about X” includes a statement of “X”.

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

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

[0105] When used herein and in the appended claims, the singular forms "a," "or," and "the" include plural subjects unless the context otherwise explicitly indicates.

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

[0107] B-cell maturation antigen (BCMA) (also known as CD269) is a member of the tumor necrosis factor receptor superfamily, namely TNFRSF17 (Thompson et al., J. Exp. Medicine, 192(1):129-135, 2000). Human BCMA is expressed almost without exception 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 activator (BAFF) and its ligand (APRIL), which is involved in proliferation (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 immunotherapies against multiple myeloma. High-affinity antibodies can block the binding of BCMA to its natural ligands, BAFF and APRIL. Anti-BCMA sdAbs can be used in combination with CAR-T cell-based cellular immunotherapy to enhance, for example, the cytotoxic effect against tumor cells.

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

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

[0110] In some embodiments, (a) a CDR1 having sequence identity with at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of an amino acid sequence selected from SEQ ID NOs: 39–76 and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. An anti-BCMA sdAb is provided comprising three CDRs, including (c) CDR2 having sequence identity of any one of %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and CDR3 having sequence identity of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with an amino acid sequence selected from SEQ ID NOs. 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 includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the anti-BCMA sdAb containing that sequence retains its ability to bind to BCMA. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 having about one, two, three, or four amino acid substitutions (e.g., conservative substitutions), insertions, or deletions to an amino acid sequence selected from SEQ ID NOs: 1-38; (b) CDR2 having about one, two, three, or four amino acid substitutions (e.g., conservative substitutions), insertions, or deletions to an amino acid sequence selected from SEQ ID NOs: 39-76; and (c) CDR3 having about one, two, three, or four amino acid substitutions (e.g., conservative substitutions), insertions, or deletions to an amino acid sequence selected from SEQ ID NOs: 77-114. In some embodiments, the anti-BCMA sdAb is affinity mature. 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 human receptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0111] In some embodiments, an anti-BCMA sdAb is provided 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, an anti-BCMA sdAb is provided 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, an anti-BCMA sdAb is provided 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, an anti-BCMA sdAb is provided 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, an anti-BCMA sdAb is provided 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, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, (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 is provided comprising three CDRs, 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 is provided comprising three CDRs, 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 is provided comprising three CDRs, 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 is provided comprising three CDRs, 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 is provided comprising three CDRs, 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 is provided comprising three CDRs, 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 sdAb is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 sdAb is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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, an anti-BCMA sdAb is provided comprising three CDRs, including (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) CDR3 comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 24, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 100.In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 25, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 26, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 27, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 28, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 66, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 104. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 29, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 30, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 106. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (a) CDR1 comprising the amino acid sequence of SEQ ID NO: 31, (b) CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and (c) CDR3 comprising the amino acid sequence of SEQ ID NO: 107. In some embodiments, an anti-BCMA sdAb is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 is provided comprising three CDRs, including (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 includes a human receptor framework, such as a human immunoglobulin framework or a human consensus framework.

[0112] In some embodiments, an anti-BCMA sdAb (i.e., an anti-BCMA sdAb including a specific CDR1, CDR2, and / or CDR3) having sequence identity of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with an amino acid sequence selected from SEQ ID NOs: 115-152. H It contains an H domain. In some embodiments, it has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity of V H The H sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-BCMA sdAb containing that sequence retains its ability to bind to BCMA. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from an amino acid sequence selected from SEQ ID NOs. 115-152. In some embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., within the FR). Optionally, an anti-BCMA sdAb contains an amino acid sequence selected from SEQ ID NOs. 115-152, including post-translational modifications of that sequence.

[0113] In some embodiments, V has an amino acid sequence selected from the group consisting of SEQ ID NOs. 115-152. H Isolated anti-BCMA sdAb containing an H domain is provided. In some embodiments, polypeptides having amino acid sequences selected from the group consisting of SEQ ID NOs: 115-152 are provided.

[0114] In some embodiments, functional epitopes can be mapped by combinatorial alanine scanning. This process uses combinatorial alanine scanning strategies to identify amino acids in the BCMA protein required for interaction with anti-BCMA sdAb. In some embodiments, the epitope can be identified using the conformation and crystal structure of the anti-BCMA sdAb bound to BCMA. In some embodiments, the present application provides BCMA epitopes derived from amino acid sequences selected from the group consisting of SEQ ID NOs: 388-394. In some embodiments, the present application provides BCMA epitopes comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 388-394.

[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 sdAbs provided herein for binding to an epitope on BCMA. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA sdAb comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, an antibody is provided that specifically binds to BCMA in competition with an anti-BCMA sdAb comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152.

[0116] In some embodiments, a competitive assay may 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 the same or sterically overlapping epitopes or by competitively inhibiting the binding of one antibody to the antigen. In certain embodiments, such a competitive antibody binds 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, NY). Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, two antibodies are said to bind to the same epitope if each blocks the binding of the other by more than 50%. In some embodiments, antibodies competing with the anti-BCMA sdAbs described herein are camelid, chimeric, humanized, or human antibodies. In some embodiments, this application provides antibodies competing 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 is provided, comprising any one of the anti-BCMA sdAbs described above. 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., VH It is an H fragment. In some embodiments, the anti-BCMA antibody is a full-length heavy-chain-only antibody containing 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 embodiments described above may incorporate, individually or in combination, any of the characteristics described in Sections 1 to 7 of the "Antibody Characteristics" section below.

[0119] In some embodiments, an isolated nucleic acid encoding one of the anti-BCMA antibodies (such as anti-BCMA sdAb) described above is provided. In some embodiments, an isolated nucleic acid encoding an anti-BCMA sdAb is provided, which comprises a sequence having at least one of the following sequence identities: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, an isolated nucleic acid comprising a nucleic acid sequence selected from the group comprising SEQ ID NOs: 153–190 is provided. In some embodiments, a vector (e.g., an expression vector) comprising such nucleic acid is provided. In some embodiments, a host cell comprising such nucleic acid is provided. In some embodiments, a method for producing an anti-BCMA antibody is provided, which comprises culturing host cells containing nucleic acids 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 concentrations 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 (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0121] In some embodiments, Kd is the Fab version or V of the antibody of interest, as described by the assay below. H It is measured by a radiolabeled antigen binding assay (RIA) performed using the H fragment and its antigen. For example, the solution binding affinity of Fab to an antibody is measured by adding Fab to the lowest concentration in the presence of a series of titrations of an unlabeled antigen. 125 I) Equilibrated with labeled antigen, and then conjugated on a plate coated with anti-Fab antibody. It is measured by capturing antibodies (see, for example, Chen et al., J.Mol.Biol.293:865-881(1999)).

[0122] In some embodiments, Kd is measured using a surface plasmon resonance assay at 25°C with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) having an immobilized antigen CM5 chip of approximately 10 response units (RUs). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RUs) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For reaction rate measurement, the Fab or V of the antibody of interest is used. H A 2-fold serial dilution of H (0.78 nM to 500 nM) is injected at a flow rate of approximately 25 μL / min into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25°C. The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorograms using a simple 1:1 Langmuir coupling 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 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the on-rate can be determined by using fluorescence quenching techniques to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band-passing) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of gradually increasing concentrations of antigen, measured with a spectrophotometer such as an Aviv Instruments spectrophotometer equipped with a stop flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) equipped with a stirring cuvette.

[0123] 2. Antibody fragments In some embodiments, the antibodies provided herein are antibody fragments. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, V H Examples include, but are not limited to, H and other fragments listed below. For an overview of a particular antibody fragment, see Hudson et al. Nat. Med. 9:129-134 (2003). For an overview of scFv, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), as well as WO93 / 16185, and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having increased in vivo half-lives, see U.S. Patent No. 5,869,046.

[0124] Diabodies are antibody fragments having two antigen-binding sites that may 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 tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

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

[0126] 3. Chimeric antibodies and humanized antibodies In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are, for example, U.S. Patent No. 4,816,567 and Morrison et al. This is described in al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody contains 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 further examples, a chimeric antibody is a "class-switched" antibody in which its class or subclass has changed from the class or subclass of the parent antibody. A chimeric antibody contains its antigen-binding fragment.

[0127] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains, where HVR, e.g., CDR (or portions thereof), is derived from a non-human antibody and FR (or portions thereof), is derived from a human antibody sequence. The humanized antibody will optionally also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues derived from a non-human antibody (e.g., an antibody from which the HVR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0128] Humanized antibodies and methods for their production are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409, and Kashmiri et al., Methods Further information is available in 36:25-34 (2005) (describes SDR(a-CDR) grafts), Padlan, Mol.Immunol.28:489-498 (1991) (describes "surface reconstruction"), Dall'Acqua et al., Methods 36:43-60 (2005) (describes "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) (describes the "inducing 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 maturation (somatic mutation) 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 (e.g., Baca et al. This includes, but is not limited to, al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0130] In some embodiments, the sdAb is modified, for example, humanized, to reduce its immunogenicity to heterologous species without reducing the domain's inherent affinity for the antigen. For example, the antibody variable domain (V) of a llama antibody. H The amino acid residues of H) can be determined, for example, by replacing one or more camelid amino acids within the framework region with their human counterparts as found in the human consensus sequence, so that the polypeptide does not lose its typical characteristics, i.e., humanization does not significantly affect the antigen-binding ability of the resulting polypeptide. Humanization of 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 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 region of the H domain is human V H It contains at least one of the following amino acid sequence homology to the framework region: approximately 50%, 60%, 70%, 80%, 90%, 95%, or more. Humanized V H One example class of the H domain is V H H is characterized by having an amino acid at position 45 (e.g., L45) from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine, according to Kabat numbering, and tryptophan at position 103. Therefore, polypeptides belonging to this class are human V H The polypeptide exhibits high amino acid sequence homology to the framework region, and can be directly administered to humans without anticipating undesirable immune responses and without burdening further humanization.

[0132] Another exemplary class of hominized camelid sdAb is described in WO03 / 035694, which is typically found in conventional antibodies of human or other species origin, but is derived from double-chain antibodies. H It contains a hydrophobic FR2 residue that compensates for this loss of hydrophilicity by substituting the conserved tryptophan residue present with a charged arginine residue at position 103. Therefore, peptides belonging to these two classes are human V H The peptide exhibits high amino acid sequence homology to the framework region, and can be directly administered to humans without expecting undesirable immune responses and without burdening further humanization.

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

[0134] Human antibodies are obtained by administering an immunogen to an intact human antibody or a transgenic animal modified to produce an intact antibody in response to antigen administration, which has a human variable region. These can be prepared by the following means. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is located extrachromosomally, or is randomly incorporated into the animal's chromosome. 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, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describes XENOMOUSE® technology), U.S. Patent No. 5,770,429 (describes HUMAB® technology), U.S. Patent No. 7,041,870 (describes KM MOUSE® technology), and U.S. Patent Application Publication No. US2007 / 0061900 (describes VELOCIMOUSE® technology). 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 hybridoma-based methods. Human myeloma and mouse-human heterozygous myeloma cell lines for the production of human monoclonal antibodies are 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 (regarding the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (regarding human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185:91 (2005).

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

[0137] V directed towards a specific antigen or target HOne technique for obtaining the H sequence is to suitably immunize a transgenic mammal capable of expressing a heavy-chain antibody (i.e., to bring about an immune response and / or a heavy-chain antibody directed against said antigen or target), and then, from said transgenic mammal containing the V H H sequence (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 V H H sequence 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 a (single) variable domain derived from a natural source (such as a human (single) variable domain, a camelid (single) variable domain, or a shark (single) variable domain), as well as any suitable (single) variable domain such as a synthetic or semi-synthetic (single) variable domain, etc.

[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 a library for antibodies having the desired binding properties are known in the art. Such methods are described, for example, by 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), a naive repertoire 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), non-rearranged V gene segments from stem cells can be cloned, highly variable CDR3 regions encoded using PCR primers containing random sequences, and a naive library synthetically generated by 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 Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

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

[0141] 6. Multispecific Antibodies In some embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. A multispecific antibody is an antibody that has binding specificity to at least two different sites. In some embodiments, one binding specificity is to an antigen selected from the group consisting of CD19, CD20, BCMA, and CD38, and the other is to any other antigen. In some embodiments, a 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 cytotoxic drugs to cells expressing an antigen selected from the group consisting of CD19, CD20, BCMA, and CD38.

[0142] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for producing bispecific antibodies include recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (Milstein and Cuello, Nature 305:537 (1983)), WO93 / 08829, and Traunecker. See et al., EMBOJ.10:3655 (1991), and the “knob-in-hole” operation (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can be produced by manipulating the electrostatic steering effect to create antibody Fc-heterodimer molecules (WO2009 / 089004A1), crosslinking 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 a leucine zipper (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)), producing bispecific antibody fragments using "diabody" technology (see, e.g., Hollinger et al., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993)), and using single-stranded Fv(sFv) dimers (see, e.g., Gruber et al. They can also be produced by preparing trispecific antibodies (e.g., Tutt et al. J.Immunol. 147:60 (1991)), and by producing polypeptides containing tandem single-domain antibodies (e.g., U.S. Patent Application No. 20110028695 and Conrath et al. J.Biol.Chem., 2001;276(10):7346-50). Manipulated antibodies having three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (e.g., see US2006 / 0025576A1).

[0143] 7. Antibody variants In some embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the antibody, and / or substitutions thereof. 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 mutants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 3. More substantial substitutions 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 are introduced into the target antibody, and the product can be screened for 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 their general side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basicity: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe.

[0146] Non-conservative substitutions involve swapping a member of one of these classes with a member of another class.

[0147] Certain types of substitutional mutants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting mutant(s) selected for further testing have a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. An exemplary substitutional mutant is an affinity-mature antibody that can be conveniently generated using phage display-based affinity maturation techniques, such as the techniques described herein. Briefly, one or more HVR residues are mutated, the mutant antibody is displayed on a phage, and it is screened for specific biological activity (e.g., binding affinity).

[0148] Modifications (e.g., substitutions) may be made within the HVR, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that undergo mutations frequently during the somatic cell maturation process (e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDR (a-CDR), resulting in mutant form V. H or V LThe binding affinity is then tested. Affinity maturation by constructing a secondary library and re-selecting from it 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 one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. This library is then screened to identify any antibody variants with 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. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Often, CDR-H3 and CDR-L3 are targeted in particular.

[0149] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within an HVR. Such modifications may be outside an HVR "hotspot" or CDR. Variant V provided above. H In some embodiments of the H sequence, each HVR is either unmodified or has one, two, or three or fewer amino acid substitutions.

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

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

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

[0153] If the antibody contains an Fc region, the carbohydrate bound to it may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides, generally bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may 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 branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of this application may be performed to produce antibody mutants having certain improved properties.

[0154] In some embodiments, antibody mutants are provided that have a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region, although Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to small sequence variations in the antibody. Such fucosylated mutants may have improved ADCC function. For example, see U.S. Patent Publication No. US2003 / 0157108 (Presta, L.) and U.S. Patent Publication No. US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications concerning "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, and WO2003 / 0845. 70. Examples include 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. Biophys. 249:533-545 (1986), US Patent Application No. US2003 / 0157108 A1 (Presta, L, and WO2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines such as knockout CHO cells of the alpha-1,6-fucosyltransferase gene, FUT8 (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 having a branched oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), US Patent No. 6,602,684 (Umana et al.), and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

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

[0157] In some embodiments, this application envisions antibody mutants that possess some, but not all, effector functions, thereby becoming desirable candidate candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion 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 therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only Fc(RIII), while 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 to evaluate the ADCC activity of target molecules are given 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 It is described in 82:1499-1502 (1985) and 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the target molecule can be measured in vivo, for example, by Clynes et al. Proc. Nat'l Acad. Sci. USA This can be evaluated in the animal models disclosed in 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. CDC assays can be performed to evaluate complement activation (e.g., Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996), Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MJ Glennie, Blood). See 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, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

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

[0159] A specific antibody variant exhibiting improved or reduced binding to FcR is 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 includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) in the Fc region.

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

[0162] Antibodies that have an extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976), and Kim et al., J.Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions therein that improve binding to FcRn in the Fc region. Such Fc variants include substitutions in 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, substitutions in the 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 Nos. 5,648,260, 5,624,821, and WO94 / 29351.

[0164] d) Cysteine-modified antibody mutants In some embodiments, it may be desirable to produce a cysteine-modified antibody, e.g., “thioMab”, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody, which can then be used to conjugate the antibody to other parts, e.g., a drug part or a linker-drug part, to produce immunoconjugates as further described herein. In some embodiments, one or more of the following residues may be substituted with cysteine: A118 (EU numbering) in the heavy chain, and S400 (EU numbering) in the heavy chain Fc region. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0165] e) Antibody derivative In some embodiments, the antibodies provided herein may be further modified to include additional non-proteinoid moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolipropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody may vary, and if one or more polymers are bound, they may be the same or different molecules. Generally, the number and / or types of polymers used in derivatization may be determined based on considerations including, but not limited to, specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under specified conditions.

[0166] In some embodiments, an antibody-nonproteinate conjugate is provided that can be selectively heated by exposure to radiation. In some embodiments, the nonproteinate portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be any wavelength, but not limited to wavelengths that do not harm normal cells, but heat the nonproteinate portion to a temperature that kills cells proximal to the antibody-nonproteinate portion.

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

[0168] The method for preparing sdAbs is described. For example, see Els Pardon et al, Nature Protocol, 2014;9(3):674. Single-domain antibody (V H H, etc., can be obtained, for example, by immunizing camelid species (such as camels or llamas) using methods known in the art and thereby 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 phage display.

[0169] For recombinant production of sdAb, the nucleic acid encoding sdAb is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding sdAb is readily isolated and sequenced using conventional methods (e.g., 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 is partly dependent on the host cell used. Generally, preferred host cells are either of prokaryotic or eukaryotic (generally mammalian) origin.

[0170] 1. Polyclonal antibody Polyclonal antibodies are generally produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and 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 1 N=C=NR (where R and R 1 are independently lower alkyl groups) can be used to conjugate the relevant antigen to a protein that is immunogenic in the immunized species, 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 the antigen, immunogenic conjugate, or derivative by intradermal injection of the solution at multiple sites, for example, by combining 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant. One month later, the animals are boost-immunized by subcutaneous injection at multiple sites with 1 / 5 to 1 / 10 of the original amount of the peptide or conjugate in complete Freund's adjuvant. Seven to fourteen days later, the animals are bled and the sera are assayed for antibody titer. 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. In addition, aggregating agents such as alum are also suitable for enhancing the immune response.

[0172] 2. Monoclonal antibody Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within that 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. Therefore, the modifier "monoclonal" indicates an antibody characteristic that it is not a mixture of distinct antibodies.

[0173] For example, monoclonal antibodies can be produced using the hybridoma method, first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA (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 induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles). and Practice, pp.59-103 (Academic Press, 1986).

[0175] Immunotherapeutic agents typically include antigen proteins or their fusion variants. Generally, peripheral blood lymphocytes ("PBLs") are used when human-derived cells are desired, or spleen cells or lymph node cells are used when non-human mammalian-derived cells are desired. The lymphocytes are then fused with immortalized cell lines using a suitable fusion 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 typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that inhibit the growth of HGPRT-deficient cells.

[0177] Preferred immortalized myeloma cells are those that efficiently fuse, support stable, high levels of antibody production by selected antibody-producing cells, and exhibit sensitivity to media such as HAT medium. Among these, those derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, as well as American Type myeloma cells, are particularly desirable. Mouse myeloma cell lines such as SP-2 cells (and their derivatives, e.g., X63-Ag8-653) available from Culture Collection, Manassas, Va. USA are preferred. Human myeloma and mouse-human heterozygous myeloma 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 antigen-directed monoclonal antibodies. Preferably, the binding specificity of the monoclonal antibodies produced by 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 the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be assayed by immunoprecipitation or in vitro binding assay, for example, radioimmunoassay. The binding affinity can be determined by radioisotope assay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the art. For example, the binding affinity can be determined by scachard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0180] After hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting dilution techniques and grown by standard methods (Goding (see above)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

[0181] Monoclonal antibodies secreted by subclones can be suitably separated from culture media, ascites fluid, or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

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

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

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

[0185] The monoclonal antibodies described herein may be monovalent, and their preparation is known in the art. For example, one method involves the recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally cleaved at any point within the Fc region to prevent heavy chain crosslinking. Alternatively, the relevant cysteine ​​residue may be substituted with another amino acid residue or deleted to prevent crosslinking. In vitro methods are also suitable for the preparation of monovalent antibodies. Digestion of the antibody to produce its fragment, specifically the Fab fragment, 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 crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutylimidate.

[0187] 3. Recombination production in prokaryotic cells a) Vector construction The polynucleotide sequences encoding the antibodies of this 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 polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the polypeptide-encoding sequences are inserted into recombinant vectors capable of replicating and expressing heterologous polynucleotides within a prokaryotic host. Many vectors available and known in the art can be used in this invention. The selection of a suitable vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cells to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cells in which it resides. Vector components generally include, but are not limited to, origins of replication, selection marker genes, promoters, ribosome-binding sites (RBS), signal sequences, heterologous nucleic acid insertions, and transcription termination sequences.

[0188] Generally, plasmid vectors containing species-derived replicons and regulatory sequences compatible with host cells are used in relation to these hosts. These vectors typically have replication sites and marking sequences that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from the E. coli species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thus providing a convenient means of identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by microorganisms 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 regulatory sequences compatible with host microorganisms can be used as transformation vectors in relation to these hosts. For example, bacteriophages such as GEM(trademark)-11 can be used to create recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0190] The expression vector of this application contains two or more promoters, each encoding a polypeptide component. --May contain cistron pairs. The promoter is a non-translational regulatory sequence located upstream (5′) of the cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes: inductive promoters and constitutive promoters. Inductive promoters are promoters that initiate transcription of increased levels of cistron under their 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. A selected promoter can be operably bound to cistron DNA encoding a 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 this application. Both native promoter sequences and many heterologous promoters can be used to induce amplification and / or expression of a target gene. In some embodiments, heterologous promoters are utilized because they generally result in greater transcription and higher yield of the expressed target gene compared to native target polypeptide promoters.

[0192] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the -lactamase and lactose promoter system, the tryptophan (trp) promoter system, and hybrid promoters, such as the tac or trc promoter. However, other promoters functional in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences are publicly available, allowing those skilled in the art to operably link them to cistrons encoding 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 embodiment, each cistron in a recombinant vector contains a secretory signal sequence component that induces transposition of the expressed polypeptide across the membrane. Generally, the signal sequence may be a component of the vector or a 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 a signal peptidase). For prokaryotic host cells that do not recognize or process the native signal sequence in heterologous polypeptides, the signal sequence is replaced, for example, with an alkaline phosphatase, penicillinase, Ipp, or a prokaryotic signal sequence selected from the group consisting of a heat-stable enterotoxin II (STII) reader, LamB, PhoE, PelB, OmpA, and MBP. In some embodiments of this application, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0194] In some embodiments, antibody production according to this application may occur in the cytoplasm of the host cell and therefore does not require the presence of a secretory signaling sequence within each cistron. In some embodiments, a polypeptide component, for example, a V of the first antigen-binding moiety which is optionally fused to the second antigen-binding moiety. HA polypeptide encoding the domain and the V of the first antigen-binding moiety which is optionally fused to the second antigen-binding moiety. L A polypeptide encoding the domain is expressed, folded, and assembled to form a functional antibody in the cytoplasm. - The company provides favorable cytoplasmic conditions for disulfide bond formation, thereby enabling proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0195] The present invention provides an expression system in which the quantitative ratio of expression polypeptide components can be adjusted to maximize the yield of secreted and appropriately assembled antibodies of this application. Such adjustment is at least partially achieved by simultaneously adjusting the translational intensity of the polypeptide components. One technique for adjusting the degree is disclosed in Simmons et al., U.S. Patent No. 5,840,523. It utilizes variants of the translation initiation region (TIR) ​​within the cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be produced at a range of translation intensities, thereby providing a convenient means for adjusting this factor to a desired expression level on a particular strand. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes that can alter the amino acid sequence, but silent changes to the nucleic acid sequence are preferred. Alterations to the TIR may include, for example, alterations to the signal sequence, as well as alterations to the number or spacing of the Shine-Dalgano sequences. One method for generating a variant signal sequence is to generate a "codon bank" at the beginning of a coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., the change is silent). This can be achieved by altering the third nucleotide position of each codon, and in addition, some amino acids such as leucine, serine, and arginine have multiple first and second positions which can complicate the bank's construction. This mutagenesis method is described in Yansura et al. (1992) METHODS: A Companion to Methods. This 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 chain, as well as the yield of the desired protein product under various TIR intensity combinations. TIR intensity is determined by Simmons As described in detail in U.S. Patent No. 5,840,523, this can be determined by quantifying the expression level of the reporter gene. Based on translation intensity comparison, the desired individual TIRs are selected to be combined in the expression vector construct of this application.

[0197] b) Prokaryotic host cells Suitable prokaryotic host cells for the expression of the antibodies of this application include Archaeobacteria 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 marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Gram-negative cells are used in some embodiments. E. coli cells are used as the host of the present invention in some embodiments. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp.1190-1219, ATCC deposit numbers 27,325) and its derivatives (genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc-fepE)degP41 kan) RExamples include strain 33D3 having the defined genotype (U.S. Patent No. 5,639,635). Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), are also preferred. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having the 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 replication potential of the replicon in bacterial cells. For example, when providing a replicon using a well-known plasmid such as pBR322, pBR325, pACYC177, or pKN410, E. coli, Serratia, or Salmonella are suitable. The species can be suitably used as a host.

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

[0199] c) Protein production Host cells are transformed with the expression vectors described above and cultured in conventional nutrient media modified to be suitable for promoter induction, transformant selection, or amplification of genes encoding desired sequences. Transformation means introducing DNA into a prokaryotic host so that the DNA can replicate either as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is carried out using standard techniques appropriate to the cell being transformed. Generally, calcium treatment using calcium chloride is used for bacterial cells that contain a substantial cell wall barrier. Another method of transformation uses polyethylene glycol / DMSO. Yet another technique used is electroporation.

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

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

[0202] Prokaryotic host cells are cultured at a suitable temperature. For E. coli growth, for example, the preferred temperature is in the range of about 20°C to about 39°C, more preferably in the range of about 25°C to about 37°C, and even more preferably about 30°C. The pH of the culture 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 inducing promoter is used in the expression vector of this application, protein expression is induced under conditions suitable for promoter activation. In one aspect of this application, the PhoA promoter is used to control polypeptide transcription. Thus, transformed host cells are cultured in phosphate-restricted medium for induction. Preferably, the phosphate-restricted medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-147). Various other inducing factors may be used according to vector constructs known and used in the art.

[0204] The expression antibody of this application is secreted into the periplasm of host cells and recovered therefrom. Protein recovery typically involves the destruction of microorganisms by means such as osmotic shock, sonication, or lysis. Once the cells are destroyed, 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 to a culture medium and isolated therein. The cells may be removed from the culture, and the culture supernatant is filtered and concentrated for further purification of the produced protein. The expression polypeptide is further isolated using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay. It can be identified.

[0205] Alternatively, protein production can be carried out on a large scale through fermentation processes. Various large-scale feed batch fermentation techniques are available for the production of recombinant proteins. Large-scale fermentation involves a capacity of at least 1,000 liters, preferably about 1,000 to 100,000 liters. These fermenters use stirring impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermenters with a volume of approximately 100 liters or less, and which may range from about 1 liter to about 100 liters.

[0206] During the fermentation process, protein expression is typically induced when the cells reach a desired density, e.g., approximately 180–220 OD. 550 Induction is initiated after the cells have grown under favorable conditions until they reach a certain stage, at which point the cells are in an early quiescent phase. Various inducing factors can be used according to the vector constructs used above that are known in the art. Cells may grow for a shorter period before induction. Cells are usually induced for about 12–50 hours, but longer or shorter induction times may also be used.

[0207] Various fermentation conditions can be modified to improve the production yield and quality of the antibodies of this application. For example, to improve the proper assembly and folding of secreted polypeptides, host prokaryotic cells can be co-transformed using a further vector overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl cis,trans-isomerase with chaperone activity). It has been demonstrated that chaperone proteins facilitate the proper folding and lysis 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 the proteolysis of expressed heterologous proteins (particularly those sensitive to proteolysis), certain host strains lacking proteolytic enzymes may be used in the present invention. For example, host cell lines may be modified to produce gene mutations(s) in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998) (see above), Georgeo et al., U.S. Patent No. 5,264,365, Georgeo et al., U.S. Patent No. 5,508,192, and Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0209] E. coli strains lacking proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins can be used as host cells in the expression system encoding the antibody of this application.

[0210] d) Purification of proteins The antibodies produced herein are further purified for further assays and use to obtain substantially identical preparations. Standard protein purification methods known in the art are used. Suitable purification procedures include: fractionation using immunoaffinity or ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography using silica or cation exchange resins such as DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

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

[0212] 4. Recombination production in eukaryotic cells In eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, and enhancer elements, promoters, and transcription termination sequences.

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

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

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

[0216] c) Selected gene components Expression and cloning vectors may contain select genes, also known as selectable markers. Typical select genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for nutritional deficiencies; or (c) supply essential nutrients unavailable from complex media, such as the gene encoding D-alanine racemase for Bacilli.

[0217] One example of a selection scheme involves using drugs that halt the growth of host cells. Cells successfully transformed using heterologous genes confine drug resistance and thus produce proteins that survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolate, and hygromycin.

[0218] Another example of a suitable selectable marker for mammalian cells is DHFR, thymidine kinase, metallothionein-I and -II, preferably the primate metallothionein gene, adenosine This enables the identification of cellular components for incorporating nucleic acids encoding the antibodies of this application, such as endoaminase and ornithine decarboxylase.

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

[0220] Alternatively, host cells transformed or co-transformed with polypeptide-coding DNA sequences, wild-type DHFR protein, and other selectable markers such as aminoglycoside 3'-phosphotransferase (APH) (in particular, wild-type hosts containing endogenous DHFR) may be selected by cell growth in a medium containing aminoglycoside antibiotics, e.g., kanamycin, neomycin, or G418, as selectors for the selectable markers. See U.S. Patent No. 4,965,199.

[0221] d) Promoter components Expression and cloning vectors typically contain a promoter that is recognized by the host organism and manipulably ligated to a nucleic acid encoding the desired polypeptide sequence. Almost all eukaryotic genes have an AT-rich region located approximately 25–30 base pairs upstream from the transcription initiation site. Another sequence found 70–80 base pairs upstream from the transcription initiation site in many genes is the CNCAAT region, where N can be any nucleotide. The 3' end of most eukaryotes is an AATAAA sequence, which can be a signal for the addition of a poly(A) 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, lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, 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-Dalgano (SD) sequence that can be manipulatively ligated to antibody-encoding DNA.

[0223] Polypeptide transcription from a vector in mammalian host cells is controlled by a heterogeneous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, or a heat shock promoter, from the genome of a virus such as polyomavirus, fowlpox virus, adenovirus (adenovirus 2, etc.), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40), provided that such a promoter is compatible with the host cell system.

[0224] Early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments that also contain the SV40 virus replication origin. The very early promoter of human cytomegalovirus can be conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papillomavirus 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. For the expression of human-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus, see also Reyes et al., Nature 297:598-601 (1982). Alternatively, the long-terminal repeat of Roussarcoma virus can be used as a promoter.

[0225] e) Enhancer component Transcription of the DNA encoding the antibody of this application by higher-order eukaryotes is often increased by inserting enhancer sequences into the vector. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are currently known. However, typically, enhancers derived from eukaryotic viruses will be used. Examples include the SV40 enhancer (bp100-270) on the posterior side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the origin of replication, and the adenovirus enhancer. For enhancing elements for eukaryotic promoter activation, see also Yaniv, Nature 297:17-18 (1982). The enhancer may be spliced ​​into the vector at the 5′ or 3′ position of the polypeptide coding sequence, but 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 region, and occasionally the 3′ untranslated region, of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments within the untranslated portion of polypeptide-coding mRNA. One useful transcription termination component is the bovine growth hormone polyadenylated region. See WO94 / 11026 and the expression vectors disclosed therein.

[0227] g) Selection and transformation of host cells Suitable host cells for cloning or expressing DNA in vectors as described herein include higher-order eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in cultures (tissue cultures) is a routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651), human embryonic kidney cell 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), and African green monkey kidney cells (VERO-76, ATCC These include CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL 51), TR1 cells (Mather et al., Annals NYAcad.Sci.383:44-68(1982)), MRC5 cells, FS4 cells, and human liver cancer cell line (Hep G2).

[0228] Host cells are transformed with the aforementioned expression or cloning vectors for antibody production and cultured in a conventional nutrient medium modified to be suitable for promoter induction, transformant selection, or amplification of genes encoding desired sequences.

[0229] h) Culture of host cells The host cells used to produce the antibodies of this application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in al., Analyst 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 may be used as culture media for host cells. Any of these media may optionally contain hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride), etc. The culture may be supplemented with minerals (such as sodium, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN®), trace elements (defined as inorganic compounds normally present at the lowest concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations that would be known to those skilled in the art. Culture conditions such as temperature and pH are those that have been used in the past with the host cells selected for expression and would be obvious to those skilled in the art.

[0230] i) Purification of proteins When using recombinant technology, antibodies can be produced intracellularly, in the periluminal lumen, or secreted directly into the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris (either host cells or lysed fragments) by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periluminal lumen of E. coli cells. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant of such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit protein degradation, and antibiotics may be included to inhibit the growth of exogenous contaminants.

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

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

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

[0234] In some embodiments, the immunoconjugate is an antibody that is a mytansinoid (U.S. Patent No. 5,208,020, U.S. Patent No. 5,416,064, and European Patent No. EP0425235) See No. B1); auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); drastatin; calicheamycin 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 See Res.58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006), Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006), Torgov et al., Bioconj. Chem. 16:717-721 (2005), Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000), Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002), King et al. See al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065, among others, which are antibody-drug conjugates (ADCs) that conjugate one or more drugs.

[0235] In some embodiments, the immunoconjugate includes, but is not limited to, antibodies described herein conjugated to an enzymatically active toxin or a fragment thereof, including diphtheria A chain, an unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, diansine protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichothecene.

[0236] In some embodiments, the immunoconjugate includes an antibody described herein that conjugates to a radioactive atom to form a radioconjugate. Various radioisotopes are available for the production of radioconjugates. For example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 Examples include radioactive isotopes of iodine, iodine, and lu. When radioactive conjugates are used for detection, they may include radioactive atoms for scintigraphy studies, e.g., tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0237] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein binders, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), 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, lysine 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 cytotoxic drugs within the cell. For example, an acid-unstable linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020).

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

[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 tissue.

[0240] In some embodiments, anti-BCMA antibodies (honmei) are used in diagnostic or detection methods. A method is provided for detecting the presence of BCMA in a biological sample (e.g., one of the anti-BCMA sdAbs described herein). In a further embodiment, a method is provided for detecting the presence of BCMA in a biological sample. In a particular embodiment, the method includes detecting the presence of the BCMA protein in a biological sample. In a particular embodiment, BCMA is human BCMA. In a particular embodiment, the method includes contacting a biological sample with an anti-BCMA antibody described herein under conditions that allow the binding of the anti-BCMA antibody to BCMA, and detecting whether a complex has formed between the anti-BCMA antibody and BCMA. Such a method may be an in vitro or 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, labeled anti-BCMA sdAb is provided. Labels include, but are not limited to, directly detectable labels or moieties (fluorescent labels, chromophore labels, electron-density labels, chemiluminescent labels, and radioactive labels, etc.), as well as moieties detected indirectly, for example, by enzymatic reactions or molecular interactions, such as enzymes or ligands. Exemplary labels include radioactive isotopes. 32 P, 14 C, 125 I, 3 H, and 131I, fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferase, such as 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, saccharido oxidase, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases coupled with enzymes that oxidize pigment precursors using hydrogen peroxide (such as HRP, lactoperoxidase, or microperoxidase), such as uricase and xanthine oxidase, biotin / avidin, spin-labeled, bacteriophage-labeled, stable free radicals, etc., are examples, but are not limited thereto.

[0242] III. Chimeric Antigen Receptors One aspect of this application relates to one or more single-domain antibodies (V H The present invention provides a chimeric antigen receptor (CAR) containing an extracellular antigen-binding domain including H, etc. Any one of the anti-BCMA sdAbs described in Section II may be used in the CAR described herein. Exemplary structures of the CAR are shown in Figures 15A-15D.

[0243] In some embodiments, a BCMA-targeting CAR (hereinafter referred to as "BCMA CAR") is provided, comprising a polypeptide including (a) an extracellular antigen-binding domain containing 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 includes a primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the BCMA CAR further comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first 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 N-terminus to 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, a BCMA CAR is provided that comprises a polypeptide including (a) an extracellular antigen-binding domain containing an anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signaling domain. sdAb includes 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: 2, CDR2 containing the amino acid sequence of SEQ ID NO: 40, and CDR3 containing the amino acid sequence of SEQ ID NO: 78; (3) 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; (4) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 42, and CDR3 containing the amino acid sequence of SEQ ID NO: 80; (5) CDR1 containing the amino acid sequence of SEQ ID NO: 5, CDR2 containing the amino acid sequence of SEQ ID NO: 43, and CDR3 containing the amino acid sequence of SEQ ID NO: 81; (6) CDR1 containing the amino acid sequence of SEQ ID NO: 6, CDR2 containing the amino acid sequence of SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 82; (7) CDR1 containing the amino acid sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 45 (8) CDR2 containing the amino acid sequence, and CDR3 containing the amino acid sequence of SEQ ID NO: 83, (9) CDR1 containing the amino acid sequence of SEQ ID NO: 8, CDR2 containing the amino acid sequence of SEQ ID NO: 46, and CDR3 containing the amino acid sequence of SEQ ID NO: 84, (10) CDR1 containing the amino acid sequence of SEQ ID NO: 9, CDR2 containing the amino acid sequence of SEQ ID NO: 47, and CDR3 containing the amino acid sequence of SEQ ID NO: 85, (11) 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, (12) 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, (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, and the amino acid sequence of SEQ ID NO: 55 CDR2, 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) amino CDR1 containing the amino acid sequence, 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 SEQ ID NO: 100 CDR3 containing the amino acid sequence, (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 SEQ ID NO: 109 (34) CDR3 containing the amino acid sequence of (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, any one of these. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or humanized. In some embodiments, the anti-BCMA sdAb is V, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. HIt includes an H domain. 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the BCMA 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 BCMA 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 comprises, from the N-terminus to the C-terminus, a CD8α signaling 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 signaling 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, BCMA CARs are provided that contain a polypeptide having sequence identity of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-256 and 298-335. Polypeptides containing amino acid sequences selected from the group consisting of SEQ ID NOs: 216-256 and 298-335 are also provided.

[0246] In some embodiments, isolated nucleic acids encoding any of the BCMA CARs provided herein are provided. In some embodiments, isolated nucleic acids are provided having sequence identity of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 257-297 and 336-373. In some embodiments, isolated nucleic acids comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 257-297 and 336-373 are 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 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 nonviral vector. Exemplary monovalent BCMA CARs are shown in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3]

[0247] Polyvalent chimeric antigen receptor This application also provides a polyvalent CAR having two or more binding sites (such as about two, three, four, five, six, or more) that specifically bind to an antigen such as BCMA. In some embodiments, one or more of the binding sites are antigen-binding fragments. In some embodiments, one or more of the binding sites include a single-domain antibody. In some embodiments, one or more of the binding sites are derived from a camelid antibody. In some embodiments, one or more of the binding sites are derived from a four-chain antibody. In some embodiments, one or more of the binding sites are scFvs. In some embodiments, one or more of the binding sites are derived from a human antibody. In some embodiments, one or more of the binding sites are polypeptide ligands or other non-antibody polypeptides that specifically bind to the antigen. In some embodiments, the polyvalent CAR is monospecific, meaning the polyvalent CAR targets a single antigen and includes two or more binding sites for that single antigen. In some embodiments, the polyvalent CAR is multispecific, meaning the polyvalent CAR targets two or more antigens and includes two or more binding sites for at least one antigen. The binding site specific to the same antigen is the same epitope of the antigen (i.e., "one epitope"). It can bind to a "tope CAR" or to different epitopes of the antigen (i.e., a "multiepitope CAR" such as two epitope CARs or three epitope CARs). Binding sites specific to the same antigen may contain the same or different sdAbs.

[0248] In some embodiments, the present application provides a polyvalent (bivalent, trivalent, or higher valency) chimeric antigen receptor comprising a polypeptide including (a) an extracellular antigen-binding domain having multiple (at least about 2, 3, 4, 5, 6 or more) binding moieties that specifically bind to an antigen (such as a tumor antigen), (b) a transmembrane domain, and (c) an intracellular signaling domain. 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 application provides a polyvalent (bivalent, trivalent, or higher valency) chimeric antigen receptor comprising a polypeptide comprising (a) an extracellular antigen-binding domain containing a plurality (at least about 2, 3, 4, 5, 6 or more) single-domain antibodies (sdAbs) that specifically bind to an antigen (e.g., 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 polyvalent (bivalent, trivalent, or higher valency) chimeric antigen receptor comprising a polypeptide including (a) an extracellular antigen-binding domain comprising a first binding moiety that specifically binds to a first epitope of an antigen (e.g., 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 and second epitopes 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 sdAb, and the second binding moiety is derived from a human antibody (e.g., scFv). In some embodiments, the first binding site is an sdAb, and the second binding site 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 polyvalent CAR specifically binds to two different epitopes on the antigen. In some embodiments, the polyvalent CAR specifically binds to three or more different epitopes on the antigen.

[0251] In some embodiments, the present application provides a polyvalent (divalent, trivalent, or higher valency) chimeric antigen receptor comprising a polypeptide comprising (a) an extracellular antigen-binding domain including 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 and second epitopes 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, sdAb(multiple sdAbs, or a first sdAb and / or The binding moieties (including a second sdAb) are camelid, chimeric, human, or humanized. In some embodiments, the binding moieties or sdAbs are fused to each other via peptide bonds or peptide linkers. In some embodiments, each peptide linker has an amino acid length of about 50 or less (e.g., 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 a primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the polyvalent CAR further includes a hinge domain (e.g., 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 polyvalent CAR further includes a signal peptide (e.g., CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the polyvalent CAR is monospecific. In some embodiments, the polyvalent CAR is multispecific, such as bispecific.

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

[0254] In some embodiments, a BCMA-targeting polyvalent CAR is provided, comprising (a) an extracellular antigen-binding domain containing multiple (at least about 2, 3, 4, or more) BCMA-binding moieties (e.g., anti-BCMA sdAbs), (b) a transmembrane domain, and (c) an intracellular signaling domain. A polyvalent BCMA CAR can be constructed using any of the anti-BCMA sdAbs. In some embodiments, the extracellular antigen-binding domain specifically binds to a single epitope of BCMA, and these CARs are referred to herein as single-epitope polyvalent BCMA CARs.

[0255] In some embodiments, a polyvalent BCMA CAR is provided comprising (a) an extracellular antigen-binding domain containing a plurality (at least about 2, 3, 4, or more) anti-BCMA sdAbs, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the anti-BCMA sdAbs include 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.

[0256] In some embodiments, a polyvalent BCMA CAR (also referred to herein as a “multiepitope polyvalent CAR”) is provided, comprising (a) an extracellular antigen-binding domain containing at least two (e.g., any one of two, three, four, 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 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 bind to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs. 388-394. In some embodiments, the first BCMA binding moiety specifically binds to an epitope derived from SEQ ID NOs. 389 and / or 390. In some embodiments, the second BCMA binding moiety specifically binds to an epitope derived from SEQ ID NOs. 391 and / or 392.

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

[0258] In some embodiments, a polyvalent BCMA CAR is provided comprising (a) an extracellular antigen-binding domain containing 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 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, and the second anti-BCMA sdAb comprises 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. In some embodiments, the first anti-BCMA sdAb comprises V containing the amino acid sequence of SEQ ID NO: 117 H It contains an H domain. In some embodiments, the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 124. H It contains an H domain. In some embodiments, the first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 124. H It contains an H domain. In some embodiments, the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 117. HIncludes the H domain.

[0259] In some embodiments, a polyvalent BCMA CAR is provided comprising (a) an extracellular antigen-binding domain containing 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 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 the anti-BCMA sdAb comprises 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. In some embodiments, the first anti-BCMA sdAb comprises V containing the amino acid sequence of SEQ ID NO: 124 H It contains an H domain. In some embodiments, the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 125. H Includes the H domain.

[0260] In some embodiments, a polyvalent BCMA CAR is provided comprising (a) an extracellular antigen-binding domain containing a first anti-BCMA sdAb and a second anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signaling domain. Ab comprises 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. Anti-BCMA sdAb comprises 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. In some embodiments, the first anti-BCMA sdAb comprises V containing the amino acid sequence of SEQ ID NO: 121. H It contains an H domain. In some embodiments, the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 125. H Includes the H domain.

[0261] In some embodiments, a polyvalent BCMA CAR is provided comprising (a) an extracellular antigen-binding domain containing 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 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, and the anti-BCMA sdAb comprises 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. In some embodiments, the first anti-BCMA sdAb comprises V containing the amino acid sequence of SEQ ID NO: 129 H It contains an H domain. In some embodiments, the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 132. H Includes the H domain.

[0262] In some embodiments, a polyvalent BCMA CAR is provided comprising (a) an extracellular antigen-binding domain containing 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 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, and the anti-BCMA sdAb comprises 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. In some embodiments, the first anti-BCMA sdAb comprises V containing the amino acid sequence of SEQ ID NO: 132 H It contains an H domain. In some embodiments, the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 134. H Includes the H domain.

[0263] In some embodiments, a polyvalent BCMA CAR is provided comprising (a) an extracellular antigen-binding domain containing 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 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, and the anti-BCMA sdAb comprises 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. In some embodiments, the first anti-BCMA sdAb comprises V containing the amino acid sequence of SEQ ID NO: 134 H It contains an H domain. In some embodiments, the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 142. H Includes the H domain.

[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 has an amino acid length of about 50 or less (e.g., one of about 35, 25, 20, 15, 10, or 5 or less). In some embodiments, the intracellular signaling domain includes the primary intracellular signaling domain of an immunoeffector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the polyvalent BCMA CAR further includes a hinge domain (e.g., 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 polyvalent BCMA CAR further includes a signal peptide (e.g., 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 signaling peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137-derived co-stimulatory signaling domain, and a CD3ζ-derived primary intracellular signaling domain. In some embodiments, the polyvalent BCMA CAR is divalent. In some embodiments, the polyvalent BCMA CAR is trivalent.In some embodiments, the polyvalent BCMA CAR specifically binds to two different epitopes on BCMA. In some embodiments, the polyvalent BCMA CAR specifically binds to three or more different epitopes on BCMA.

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

[0266] In some embodiments, isolated nucleic acids encoding any of the polyvalent BCMA CARs provided herein are provided. In some embodiments, isolated nucleic acids are provided having sequence identity of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 336-373. In some embodiments, isolated nucleic acids comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 336-373 are 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 any of the polyvalent 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 nonviral vector. Exemplary polyvalent BCMA CARs are shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0267] Multiple specific chimeric antigen receptors This application further provides multispecific chimeric antigen receptors that target two or more different antigens (such as one of about 2, 3, 4, 5, 6, or more). In some embodiments, the multispecific CAR has one antigen-binding site for each antigen. In some embodiments, the multispecific CAR has two or more binding sites for at least one antigen. Each antigen-binding site may contain an sdAb. For example, in some embodiments, the multispecific CAR is a bispecific CAR that includes an extracellular antigen-binding domain containing two different sdAbs that each specifically bind to an antigen. In some embodiments, multiple A specific CAR is a triple-specific CAR that contains an extracellular antigen-binding domain, each containing three different sdAbs that specifically bind to different antigens.

[0268] In some embodiments, a polyspecific (bispecific) chimeric antigen receptor (CAR) is provided, comprising a polypeptide including (a) an extracellular antigen-binding domain containing 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), (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the first antigen is different from the second antigen. In some embodiments, the second antigen is 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 are 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 has an amino acid length of about 50 or less (e.g., 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 (e.g., 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the multispecific CAR further includes a hinge domain (e.g., 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 located at the N-terminus of the polypeptide (e.g., CD8α signal peptide). In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137-derived co-stimulatory signaling domain, and a CD3ζ-derived primary intracellular signaling domain. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a CD28-derived co-stimulatory signaling domain, and a CD3ζ-derived primary intracellular signaling domain.

[0269] Extracellular antigen-binding domain The extracellular antigen-binding domains of CARs described herein include one or more binding sites (such as one, two, three, four, five, six, or more) such as sdAb. In some embodiments, one or more binding sites are antibodies or their antigen-binding fragments. In some embodiments, one or more binding sites are derived from four-chain antibodies. In some embodiments, one or more binding sites are derived from camelid antibodies. In some embodiments, one or more binding sites are derived from human antibodies. In some embodiments, one or more binding sites are non-antibody-binding proteins, such as polypeptide ligands or engineered proteins that bind to antigens. The binding sites may be directly fused to one another via peptide bonds or peptide linkers.

[0270] 1. Single-domain antibody In some embodiments, the CAR is an extracellular antigen-binding domain containing one or more sdAbs. It contains. sdAbs may be of the same or different origin and may be of the same or different size. Exemplary sdAbs include heavy chain only antibodies (e.g., V H H or V NAR ) Heavy chain variable domain from, binding molecule that naturally lacks a light chain, single domain (V) derived from conventional 4-chain antibodiesH or V L Examples include, but are not limited to, humanized heavy chain-only antibodies, human sdAbs produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single-domain scaffolds other than those derived from antibodies. The CARs described herein can be constructed using any sdAb known in the art or developed by the inventors, including the sdAbs described in Section II of this application. The sdAbs may be derived from any species, including but not limited to mice, rats, humans, camels, llamas, lampreys, fish, sharks, goats, rabbits, and cattle. The single-domain antibodies contemplated herein also include naturally occurring sdAbs from species other than camelids and sharks.

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

[0272] V from the camelid family H H molecules are about 10 times smaller than IgG molecules. They are single polypeptides, very stable, and can withstand extreme pH and temperature conditions. Furthermore, they can withstand protease activity, which is not the case with conventional 4-chain antibodies. HIn vitro expression of H yields high yield of properly folded functional V H This results in H. In addition, antibodies produced in camelids can recognize epitopes other than those recognized by antibodies produced in vitro, either through the use of antibody libraries or through immunization of non-camelid mammals (see, for example, WO9749805). Therefore, one or more V H Multispecific or multivalent CARs containing an H domain can interact with targets more efficiently than multispecific or multivalent CARs containing antigen-binding fragments derived from conventional four-chain antibodies. H Since H is known to bind to "abnormal" epitopes such as cavities or grooves, such V H The affinity of CARs containing H may be more favorable for therapeutic treatment than that of conventional multispecific polypeptides.

[0273] In some embodiments, sdAb is derived from the variable region of immunoglobulins found in cartilaginous fish. For example, sdAb may be derived from an immunoglobulin isotype known as a novel antigen receptor (NAR) found in shark serum. Methods for producing single-domain molecules derived from the variable region of NARs ("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 in vitro generated (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework region may be modified by "camelization" of specific amino acid residues within the framework region. Camelization is performed using (naturally occurring) V from conventional 4-chain antibodies. H V of heavy chain antibody, one or more amino acid residues in the amino acid sequence of the domain H It is produced at the corresponding location(s) within the H domain. This refers to the substitution or replacement of one or more amino acid residues. This may be done in a manner that is inherently known, for example, based on further description herein. Such “camelization” substitution is preferably V H -V L They form an interface and / or are inserted into amino acid positions present at that interface and / or into so-called camelid characteristic residues as defined herein (e.g., WO94 / 04678, Davies and See 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 expressing 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, naturally occurring V against a specific antigen or target H The H domain is found in Camelidae V HThe H sequence may be obtained from a (natural or immunoassay) library. Such a method may or may not involve screening such library using the antigen or target, or at least one part, fragment, antigenic determinant, or epitope thereof, using one or more screening techniques that are originally known. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, the H sequence may be obtained from a (natural or immunoassay) library by techniques such as random mutagenesis and / or CDR shuffling as described, for example, in WO00 / 43507. H V obtained from the H Library H (natural or immunoviral) V from H Library etc. H Improved synthetic or semi-synthetic libraries derived from the H library may be used.

[0277] In some embodiments, the sdAb is generated from a conventional four-chain antibody. For example, EP See 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 this application are cell surface molecules. The binding moiety (e.g., sdAb) may be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a specific disease condition. In some embodiments, the antigen(s) (e.g., a first antigen and / or a second antigen) are tumor antigens. 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(s) targeted by the CAR may be an antigen on a single diseased cell or an antigen expressed on different cells, each contributing to the disease. The antigen(s) targeted by the CAR may 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 targeted antigens in this invention will depend on the specific type of cancer being treated. Exemplary tumor antigens include, for example, glial antigens. Examples include tumor-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostain, PSMA, HER2 / neu, sulbibin 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, tumor antigens comprise one or more antigenic oncological epitopes associated with malignant tumors. Malignant tumors express several proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens such as carcinoembryonic antigens (CEAs). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphoma.

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

[0282] Non-limiting examples of TSA or TAA antigens include: differentiation antigens, e.g., MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multiseries antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutant tumor suppressor genes, e.g., p53, Ras, HER2 / neu; specific tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and viral antigens, e.g., Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\Cyclophyllin C-related protein, TAAL6 Examples include TAG72, TLP, and TPS.

[0283] In some embodiments, the antigen (first antigen and / or 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 Various binding sites (e.g., sdAbs) in the multispecific or multivalent CARs described herein can be fused to one another via peptide linkers. In some embodiments, the binding sites (e.g., sdAbs) are fused directly to one another without any peptide linkers. The peptide linkers linking different binding sites (e.g., sdAbs) may be the same or different. Different domains of the CAR can also be fused to one another via peptide linkers.

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

[0286] The peptide linker may be of any preferred length. In some embodiments, the peptide linker has an amino acid length of 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. In some embodiments, the peptide linker has an amino acid length of 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 less. In some embodiments, the length of the peptide linker is one of the following: approximately 1 amino acid to approximately 10 amino acids, approximately 1 amino acid to approximately 20 amino acids, approximately 1 amino acid to approximately 30 amino acids, approximately 5 amino acids to approximately 15 amino acids, approximately 10 amino acids to approximately 25 amino acids, approximately 5 amino acids to approximately 30 amino acids, approximately 10 amino acids to approximately 30 amino acids, approximately 30 amino acids to approximately 50 amino acids, approximately 50 amino acids to approximately 100 amino acids, or approximately 1 amino acid to approximately 100 amino acids.

[0287] Peptide linkers may have sequences that are naturally occurring or sequences that are not naturally occurring. For example, sequences derived from the hinge region of heavy-chain only antibodies can be used as linkers. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. An example of a flexible linker is a glycine polymer (G). n , glycine-serine polymer (e.g., (GS) n (GSGGS) n (GGGS) n , and (GGGGS) n (including, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other polymers known in the art. Flexible linkers are one example. In some embodiments, the peptide linker includes the amino acid sequence GGGGS (SEQ ID NO: 208), (GGGGS)2 (SEQ ID NO: 209), (GGGS)4 (SEQ ID NO: 210), GGGGSGGGGSGGGGGGSGSGGGGS (SEQ ID NO: 211), GGGGSGGGGSGGGGGGSGSGGGGGGSGGGGGS (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 this application include a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain may be derived from either a natural or synthetic source. As used herein, “transmembrane domain” refers to any protein structure that is thermodynamically stable within the cell membrane, preferably within the eukaryotic cell membrane. A transmembrane domain suitable for use in the CARs described herein may be obtained from naturally occurring proteins. Alternatively, it may be a synthetic, non-naturally occurring protein segment, such as a thermodynamically stable hydrophobic protein segment within the 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 cellular phospholipid bilayer. In addition, transmembrane domains can be classified, or alternatively, based on their transmembrane domain topology, which includes the number of times the transmembrane domain crosses the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, while multi-pass membrane proteins cross the cell membrane at least twice (e.g., two, three, four, five, six, seven, or more times). Membrane proteins can be defined as type I, type II, or type III depending on the topology of their terminals and membrane-crossing segments (may be multiple) relative to the inside and outside of the cell. Type I membrane proteins have a single-membrane-crossing region and are oriented so that the N-terminus of the protein lies extracellularly on the cellular lipid bilayer and the C-terminus of the protein lies cytoplasmically. Type II membrane proteins also have a single-membrane region, but their C-terminus is oriented to be on the extracellular side of the cell's lipid bilayer, while their N-terminus is oriented to the cytoplasm. Type III membrane proteins have segments that span multiple membranes and can be further subdivided based on the number of transmembrane segments and the positions of their N-terminus and C-terminus.

[0290] In some embodiments, the transmembrane domains of the CARs described herein are derived from type I single-pass membrane proteins. 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 may comprise a complex (at least 2, 3, 4, 5, 6, 7, or more) alpha-helix or beta-sheet structure. Preferably, the N-terminus and C-terminus of the multi-pass membrane protein are located 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 CAR is the transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The molecule includes a transmembrane domain selected from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (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 containing 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 containing 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 domains for use in CARs described herein may also include at least a portion of a synthetic, naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, naturally occurring alpha-helix or beta-sheet. In some embodiments, the protein segment consists of at least approximately 20 amino acids, e.g., 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, e.g., U.S. Patent No. 7,052,906 B1 and PCT Publication No. WO2000 / 032776 A2, and these relevant disclosures are incorporated herein by reference.

[0295] The transmembrane domain may include a transmembrane region and a cytoplasmic region located at the C-terminal end of the transmembrane domain. The cytoplasmic region of the transmembrane domain may contain three or more amino acids, which in some embodiments help 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 contains positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain contains amino acids, arginine, serine, and lysine.

[0296] In some embodiments, the transmembrane region of the transmembrane domain contains hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the CAR contains 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 contains 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 contains a polyleucine-alanine sequence. The hydroxyl or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, such as Kite and Doolittle's hydroxyl analysis.

[0297] Intracellular signal transduction domains The CAR of this application includes an intracellular signaling domain. The intracellular signaling domain is responsible for the activation of at least one of the normal effector functions of an immunoeffector cell expressing the CAR. The term “effector function” refers to a cellular specialization function. For example, the effector function of a T cell may be helper activity, including cytolytic activity or cytokine secretion. Therefore, the term “cytoplasmic signaling domain” refers to the portion of a protein that transduces the effector function signal and instructs the cell to perform the specialization function. Usually, the entire cytoplasmic signaling domain may be used, but in many cases, it is not necessary to use the entire chain. To the extent that a cleavage portion of the cytoplasmic signaling domain is used, such cleavage portion may be used in place of the intact chain, insofar as it transduces the effector function signal. Therefore, the term cytoplasmic signaling domain is intended to include any cleavage portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.

[0298] In some embodiments, the intracellular signaling domain includes the primary intracellular signaling domain of the immune effector cell. In some embodiments, the CAR includes an intracellular signaling domain that essentially consists of the primary intracellular signaling domain of the immune effector cell. "Primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulating manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immune receptor tyrosine-based activation motif or 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 may consist of two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is any amino acid that independently yields the conserved motif YxxL / Ix(6-8)YxxL / I. The ITAM within the 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 may 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 contains 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 containing one or more mutations, such as Q65K. In some embodiments, the primary intracellular signaling domain of mutant CD3ζ contains 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 antigen-specific signaling to promote cell proliferation, differentiation, and survival, and to activate the cell's effector function. In some embodiments, CARs include at least one co-stimulatory signaling domain. As used herein, the term “co-stimulatory signaling domain” refers to an intracellular signatory signaling domain. This refers to at least a portion of a protein that induces an immune response, such as effector function, by mediating transduction. The co-stimulatory signaling domain of the chimeric receptor described herein may be a cytoplasmic signaling domain from a co-stimulatory protein that transduces signals and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The “co-stimulatory signaling domain” may be the cytoplasmic portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a congenital 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 an immune cell, including but not limited to proliferation and survival.

[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 (approximately two, three, four, or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same co-stimulatory signaling domains, 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 derived from different co-stimulatory proteins, such as two or more 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, 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 an arbitrary peptide linker. The primary intracellular signaling domain and one or more co-stimulatory signaling domains may be arranged in any preferred order. In some embodiments, 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-stimulus signaling domains may provide additive or synergistic stimuli.

[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 CARs as described herein. The type(s) of the co-stimulatory signaling domain is selected based on factors such as the type of immune effector cell on which the effector molecule is expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). Examples of co-stimulatory signaling domains for use in CAR include members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6), and 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 TNFRII / TNFRSF1B), members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150), as well as any other co-stimulatory molecules, e.g., CD2, CD7, CD53, CD82 / Kai-1, This may include, but is not limited to, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin alpha-4 / CD49d, integrin alpha-4 beta-1, integrin alpha-4 beta-7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C, as well as cytoplasmic signaling domains of costimulatory proteins.

[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 this application includes a co-stimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain includes a 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 this application includes a co-stimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain includes a 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 this application includes a CD28 co-stimulatory signaling domain and a CD137 co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain includes a CD3ζ cytoplasmic signaling domain, a CD28 co-stimulatory signaling domain, and a CD137 co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain includes, from the N-terminus to the C-terminus, a CD28 co-stimulatory signaling domain, a CD137 co-stimulatory signaling domain, and a CD3ζ cytoplasmic signaling domain. In some embodiments, the CD28 co-stimulatory signaling domain includes the amino acid sequence of SEQ ID NO: 195. In some embodiments, the CD137 co-stimulatory signaling domain includes the amino acid sequence of SEQ ID NO: 196.

[0307] Since the co-stimulatory signaling domains can modulate the immune response of immune cells, any variant of the co-stimulatory signaling domains described herein is also within the scope of this disclosure. In some embodiments, the co-stimulatory signaling domains include up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to the wild-type counterpart. Such co-stimulatory signaling domains including one or more amino acid variations may be referred to as variants. Amino acid residue variations in a co-stimulatory signaling domain are considered to be co-stimulatory signals that do not include the mutation. Compared to the signaling domain, this can result in increased signal transduction and enhanced stimulation of the immune response. Mutations in amino acid residues of the co-stimulatory signaling domain can result in decreased signal transduction and reduced stimulation of the immune response compared to the non-mutated co-stimulatory signaling domain.

[0308] Hinge area The CAR of this application may include a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is generally an amino acid segment found between two domains of a protein, which can allow for the flexibility of the protein and the movement of one or both of those domains toward each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain toward the transmembrane domain of the effector molecule may be used.

[0309] The hinge domain may contain approximately 10 to 100 amino acids, for example, one of approximately 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain may be at least one of approximately 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 acid lengths.

[0310] In some embodiments, the hinge domain is a naturally occurring hinge domain of a protein. Any hinge domain of a protein known in the art for containing a hinge domain is suitable for use in the chimeric receptor described herein. In some embodiments, the hinge domain is at least a portion of a naturally occurring hinge domain of a protein that confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, for example, a fragment of the hinge domain of CD8α containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids. In some embodiments, the hinge domain of CD8α contains 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 a hinge domain that links the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge domain is of an antibody that includes the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain includes the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain includes 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 includes the hinge region of the IgG1 antibody and the CH2 and CH3 constant regions. In some embodiments, the hinge region includes the hinge region of the IgG1 antibody and the constant CH3 region.

[0312] Peptides that do not exist in nature may also be used as hinge domains of the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of the Fc receptor is a peptide linker, for example, a (GxS)n linker, where x and n can independently be integers from 3 to 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0313] Signal peptide The CARs of this application may include a signal peptide (also known as a signal sequence) at the N-terminus of a polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets an effector molecule to a cellular secretory pathway, enabling the integration and anchoring of the effector molecule to the lipid bilayer. Signal peptides, including signal sequences of naturally occurring proteins or synthetic non-natural signal sequences, that are suitable for use in the CARs described herein will be apparent to those skilled in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the signal peptide of CD8α includes 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. Manipulated immune effector cells A host cell (such as an immune effector cell) containing any one of the CARs described herein is further provided in this application.

[0315] Accordingly, in some embodiments, engineered immune effector cells (such as T cells) are provided that include a polyvalent CAR comprising a polypeptide comprising (a) an extracellular antigen-binding domain including 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 and second epitopes are different.

[0316] In some embodiments, an engineered immunoeffector cell (such as a T cell) is provided which contains a polyvalent CAR comprising a polypeptide comprising (a) an extracellular antigen-binding domain including a first anti-BCMA sdAb that specifically binds to a first epitope of BCMA and a second anti-BCMA sdAb that specifically binds to a second epitope of BCMA, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the first and second epitopes are different. In some embodiments, the first anti-BCMA sdAb and / or the second anti-BCMA sdAb are camelid, chimeric, human, or humanized. In some embodiments, the first and second anti-BCMAs are fused to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker has an amino acid length of about 50 or less (e.g., 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 (e.g., 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the polyvalent CAR further includes a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the polyvalent CAR further comprises a signal peptide located at the N-terminus of the polypeptide (e.g., CD8α signal peptide).In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signaling peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137-derived co-stimulatory signaling domain, and a CD3ζ-derived primary intracellular signaling domain. 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, engineered immune effector cells (such as T cells) are provided that contain a polypeptide comprising (a) an extracellular antigen-binding domain containing an anti-BCMA sdAb, (b) a transmembrane domain, and (c) an intracellular signaling domain, and anti-BCMA sdAb includes 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: 2, CDR2 containing the amino acid sequence of SEQ ID NO: 40, and CDR3 containing the amino acid sequence of SEQ ID NO: 78; (3) 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; (4) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 42, and CDR3 containing the amino acid sequence of SEQ ID NO: 80; (5) CDR1 containing the amino acid sequence of SEQ ID NO: 5, CDR2 containing the amino acid sequence of SEQ ID NO: 43, and CDR3 containing the amino acid sequence of SEQ ID NO: 81; (6) CDR1 containing the amino acid sequence of SEQ ID NO: 6, CDR2 containing the amino acid sequence of SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 82; (7) CDR3 containing the amino acid sequence of SEQ ID NO: 7 1. CDR2 containing the amino acid sequence of SEQ ID NO: 45, and CDR3 containing the amino acid sequence of SEQ ID NO: 83, (8) CDR1 containing the amino acid sequence of SEQ ID NO: 8, CDR2 containing the amino acid sequence of SEQ ID NO: 46, and CDR3 containing the amino acid sequence of SEQ ID NO: 84, (9) CDR1 containing the amino acid sequence of SEQ ID NO: 9, CDR2 containing the amino acid sequence of SEQ ID NO: 47, and CDR3 containing the amino acid sequence of SEQ ID NO: 85, (10) 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, and 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) the amino acid sequence of SEQ ID NO: 17 (18) CDR1 containing the amino acid sequence of SEQ ID NO: 55, CDR2 containing the amino acid sequence of SEQ ID NO: 93, (19) CDR1 containing the amino acid sequence of SEQ ID NO: 19, CDR2 containing the amino acid sequence of SEQ ID NO: 57, 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 the amino acid sequence of SEQ ID NO: 96 CDR3 containing the sequence, (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, SEQ ID NO: 62 CDR2 containing the amino acid sequence of (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, and CDR3 containing the amino acid sequence of SEQ ID NO: 71 (34) CDR3 containing the amino acid sequence of R2 and SEQ ID NO: 109, (35) 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, (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 extracellular antigen-binding domain contains at least two anti-BCMA sdAbs. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or humanized. In some embodiments, the anti-BCMA sdAb is V, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. HIt includes an H domain. 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 ligands for CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the BCMA 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 BCMA 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 comprises, from N-terminus to C-terminus, a CD8α signaling 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 N-terminus to C-terminus, a CD8α signaling 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-256 and 298-335. In some embodiments, the engineered immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoi...

Claims

1. A polypeptide-containing chimeric antigen receptor (CAR), (a) An extracellular antigen-binding domain comprising a first BCMA-binding moiety and a second BCMA-binding moiety, wherein the first BCMA-binding moiety is a first anti-BCMA single-domain antibody (sdAb), and the second BCMA-binding moiety is a second anti-BCMA sdAb, (b) Transmembrane domain and (c) an intracellular signaling domain, The first anti-BCMA sdAb and the second anti-BCMA sdAb are, independently of each other, (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: 2, CDR2 containing the amino acid sequence of SEQ ID NO: 40, and CDR3 containing the amino acid sequence of SEQ ID NO:

78. (3) 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 (4) CDR1 containing the amino acid sequence of SEQ ID NO: 4, CDR2 containing the amino acid sequence of SEQ ID NO: 42, and CDR3 containing the amino acid sequence of SEQ ID NO:

80. (5) CDR1 containing the amino acid sequence of SEQ ID NO: 5, CDR2 containing the amino acid sequence of SEQ ID NO: 43, and CDR3 containing the amino acid sequence of SEQ ID NO:

81. (6) CDR1 containing the amino acid sequence of SEQ ID NO: 6, CDR2 containing the amino acid sequence of SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO:

82. (7) 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 (8) CDR1 containing the amino acid sequence of SEQ ID NO: 8, CDR2 containing the amino acid sequence of SEQ ID NO: 46, and CDR3 containing the amino acid sequence of SEQ ID NO:

84. (9) CDR1 containing the amino acid sequence of SEQ ID NO: 9, CDR2 containing the amino acid sequence of SEQ ID NO: 47, and CDR3 containing the amino acid sequence of SEQ ID NO: 85 (10) 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, and the amino acid sequence of SEQ ID NO: 58 CDR2 containing the amino acid sequence of SEQ ID NO: 96, 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) 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, (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 The chimeric antigen receptor (CAR) is an sdAb that includes any of the following selected from the group consisting of the following.

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