BR122024003787A2 - CHIMERIC ANTIGEN RECEPTORS THAT TARGETING BCMA AND METHODS OF USE THEREOF - Google Patents

Abstract

The present application provides single-domain antibodies that target BCMA and chimeric antigen receptors (such as monovalent CAR and multivalent CAR including biepitope CAR) comprising one or more anti-BCMA single-domain antibodies. Additionally, engineered immune effector cells (such as T cells) that comprise chimeric antigen receptors are provided. Pharmaceutical compositions, kits and cancer treatment methods are also provided.

Description

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

PRESENTATION OF SEQUENCE LISTING IN ASCII TEXT FILE

[0002] The content of the following ASCII text file presentation is incorporated herein by reference in its entirety: a machine readable form (CRF) of the Sequence Listing (file name: 761422000640SEQLISTING. txt, registered date: August 10, 2017, size: 520 KB).

FIELD OF THIS REQUEST

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

BASIS OF THIS REQUEST

[0004] With the development of tumor immunotherapy and clinical technology, chimeric antigen receptor T cell (CAR-T) immunotherapy is now one of the most promising tumor immunotherapy approaches. With the development of immunotherapy and medical technology, chimeric antigen receptor (CAR) T cell immunotherapy is now one of the most promising tumor immunotherapy approaches. The extracellular antigen-binding domain may comprise a single-chain variable fragment (scFv) targeting an identified tumor antigen. CARs can be expressed on the surface of T cells using gene transfection techniques. Upon binding to target tumor antigen, CARs can activate T cells to initiate specific antitumor response in an antigen-dependent manner without being limited by the availability of major histocompatibility complexes (MHC) specific to the target tumor antigen.

[0005] Single domain antibodies (sdAbs) are different from conventional 4-chain antibodies by having a single monomeric antibody variable domain. For example, camelid and shark sdAbs called heavy chain antibodies (HcAbs), which naturally lack light chains. The antigen-binding fragment in each arm of camelid heavy chain antibodies has a heavy chain variable domain (VHH), which can have high affinity for an antigen without the assistance of a light chain. Camelid VHH is known as the smallest functional antigen-binding fragment with a molecular weight of approximately 15 kD.

[0006] Multiple myeloma (MM) is an incurable aggressive plasma malignancy, which is categorized as a B-cell neoplasm and proliferates uncontrollably in the bone marrow, interfering with the normal metabolic production of blood cells and causing painful bone lesions (Garfall, A. L. et al., Discovery Med. 2014, 17, 37). Multiple myeloma can present clinically with hypercalcemia, renal failure, anemia, bone lesions, bacterial infections, hyperviscosity, and amyloidosis (Robert Z. Orlowski, Cancer Cell. 2013, 24(3)). According to research and statistics, about 86,000 patients will be diagnosed each year with myeloma, and about 63,000 patients die every year from complications related to the disease (Becker, 2011). Because of the aging population, the number of myeloma cases is predicted to increase year by year. Like many types of cancer, there is no known cause of multiple myeloma and no cure. Some treatments for multiple myeloma are similar to treatments for other types of cancer, such as chemotherapy or radiation therapy, stem cell transplant or bone marrow transplant, targeted therapy, or biological therapy (George, 2014). Antibody-based cell immunotherapies have demonstrated substantial clinical benefits for patients with hematologic malignancies, particularly in B-cell non-Hodgkin lymphoma. Although current therapies for multiple myeloma lead to remissions, nearly all patients eventually relapse. There is a need for an effective immunotherapeutic agent to treat multiple myeloma.

[0007] LCAR-B38M disclosed in the present invention is a bivalent BCMA that targets CAR-T and has already demonstrated clinical advantages in terms of safety and efficacy in the treatment of patients with refractory or relapsed multiple myeloma in a clinical trial. In an initial clinical trial, 33 of 35 (94%) patients experienced clinical remission of multiple myeloma when receiving LCAR-B38M CAR T-cells. Most patients had only mild side effects. The study was presented by the lead inventor at the 2017 ASCO annual meeting (Abstract LBA3001) and press conference that garnered widespread media coverage. (https://www.ascopost.com/News/55713).

[0008] Overall, the objective response rate was 100% and 33 patients (94%) had an evident clinical remission of myeloma (complete response, very good partial response, or partial response) within 2 months of receiving the cells T CAR. After following the group for a period of more than 4 months, of the 19 patients, 14 achieved strict complete response criteria, 1 patient achieved partial response and 4 patients achieved very good partial remission criteria in efficacy.

[0009] Because the excellent efficacy and safety profile obtained from the LCAR-B38M clinical study is significantly superior to that of some other BCMA CAR-T studies reported at the same time at ASCO, the work was widely recognized as a “revolutionary advance” in field of immunotherapy. It is notable that all of these BCMA CAR designs are conventional CARs in which the BCMA antigen-binding domain is composed of a monovalent ScFv antibody.

[0010] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THIS APPLICATION

[0011] The present application provides anti-BCMA single domain antibodies (sdAb), chimeric antigen receptors (CARs) comprising one or more anti-BCMA sdAbs (such as VHH fragments), engineered immune effector cells, and methods of using the same in cancer immunotherapy.

[0012] One aspect of the present application provides an anti-BCMA sdAb comprising the CDR regions of any of SEQ ID NOs:115-152. In some embodiments, the anti-BCMA sdAb comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52; and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114. In some embodiments, the anti-BCMA sdAb comprises a VHH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:115-152.

[0013] In some embodiments, an anti-BCMA heavy chain antibody (HCAB) or an antigen-binding protein comprising any of the anti-BCMA sdAbs described above is provided. Also provided are BCMA epitopes that any of the anti-BCMA sdAbs described above specifically bind to anti-BCMA antibodies (such as anti-BCMA sdAbs) that compete with any of the anti-BCMA sdAbs described above.

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

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

[0016] One aspect of the present application provides a multivalent chimeric antigen receptor (CAR) comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety and a second BCMA-binding moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, one or more of the first BCMA-binding moiety and the second BCMA-binding moiety is an anti-BCMA sdAb. In some embodiments, the first BCMA-binding moiety is a first anti-BCMA sdAb and the second BCMA-binding moiety is a second anti-BCMA sdAb. In some embodiments, the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is derived from a human antibody. In some embodiments, the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is a BCMA polypeptide ligand. In some embodiments, the first BCMA-binding moiety and the second BCMA-binding moiety specifically bind to the same epitope on BCMA. In some embodiments, the first BCMA-binding moiety and the second BCMA-binding moiety specifically bind to different epitopes on BCMA. In some embodiments, the first BCMA-binding moiety and/or the second BCMA-binding moiety specifically binds to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs:388-394. In some embodiments, the first BCMA-binding moiety specifically binds to an epitope derived from SEQ ID NO:389 and/or 390. In some embodiments, the second BCMA-binding moiety specifically binds to an epitope derived from SEQ ID NO:391 and/or 392.

[0017] One aspect of the present application provides a multivalent (such as bivalent or trivalent) chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb (such as any of the sdAbs anti-BCMA described above) and a second anti-BCMA sdAb (such as any of the anti-BCMA sdAbs described above); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the first anti-BCMA sdAb and the second anti-BCMA sdAb specifically bind to the same epitope on BCMA. In some embodiments, the first anti-BCMA sdAb and the second anti-BCMA sdAb specifically bind to different epitopes on BCMA. In some embodiments, the first anti-BCMA sdAb and/or the second anti-BCMA sdAb specifically binds to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs:388-394. In some embodiments, the first anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NO: 389 and/or 390. In some embodiments, the second anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NO: 391 and/or 392.

[0018] In some embodiments according to any of the multivalent CARs provided above, the first BCMA-binding moiety (e.g., the first anti-BCMA sdAb) is located at the N-terminus of the second BCMA-binding moiety (e.g., example, 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 together via a bond. peptide. 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 linker. peptide. In some embodiments, the peptide linker is no more than about 50 (such as no more than 35, 25, 20, 15, 10, or 5) amino acids in length. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs:208-215.

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

[0020] In some embodiments according to any of the CARs (including multivalent CARs) described above, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3α. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 197 or 198.

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

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

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

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

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

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

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

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

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

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGS. 1A-1B show the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific CARs comprising various anti-BCMA sdAbs against RPMI8226.Luc cells (FIG. 1A), or U87MG.Luc cells (FIG. 1B).

[0033] FIGS. 2A-2C show the results of an in vitro cytotoxicity assay of T cells expressing exemplary monospecific CARs comprising various anti-BCMA sdAbs against RPMI8226.Luc cells (FIG. 2A), K562.BCMA.Luc cells (FIG. 2B), or K562.CD19.Luc cells (FIG. 2C).

[0034] FIG. 3 shows results of an in vitro IFNY release assay from T cells expressing exemplary monospecific CARs comprising various anti-BCMA sdAbs against K562.BCMA.Luc cells.

[0035] FIGS. 4A-4C show the results of an in vitro cytotoxicity assay of T cells expressing exemplary multivalent BCMA CARs against RPMI8226.Luc cells (FIGS. 4A-4B) or U87MG.Luc cells (FIG. 4C).

[0036] FIGS. 5A-5E show the results of an in vitro cytotoxicity assay of T cells expressing exemplary bivalent BCMA CARs against RPMI8226.Luc cells (FIG. 5A), K562.CD19.Luc cells (FIG. 5B), A549.Luc cells (FIG. 5B), FIG. 5C), U87MG.Luc cells (FIG. 5D) or Raji.Luc cells (FIG. 5E).

[0037] FIG. 5F shows results of an in vitro cytotoxicity assay of exemplary bivalent BCMA CAR-expressing T cells against K562.BCMA.Luc cells and K562 cells. CD38. Luc.

[0038] FIG. 6A shows results of an in vitro IFNγ release assay from T cells expressing exemplary bivalent BCMA CARs against K562.BCMA.Luc cells at two different effector cell to target cell ratios.

[0039] FIG. 6B shows the results of an in vitro IFNγ release assay from T cells expressing exemplary bivalent BCMA CARs against RPMI8226.Luc, A549.Luc, K562 cells. CD38. Luc and Raji.Luc.

[0040] Figures 7A-7C show the binding of three exemplary VHH fragments to K562.BCMA.Luc cells and K562 cells. CD38. Luc (negative control).

[0041] FIG. 8A shows a crystal structure of the extracellular domain of BCMA. FIG. 8B shows BCMA epitope peptides.

[0042] FIGs 9A-9B show results of VHH1 and VHH2 epitope mapping assays.

[0043] FIG. 10 shows results from a competitive binding assay using CHO-BCMA cells.

[0044] FIG. 11 shows in vitro cytotoxicity of donor-derived T cells expressing LCAR-B38M against RPMI8226.Luc cells

[0045] FIG. 12A shows the in vitro cytotoxicity of LCAR-B38M CAR T-cells prepared from a selected donor against RPMI8226 cells. 12B-12E shows the in vivo antitumor activity of LCAR-B38M T-CAR cells in a tumor xenograft mouse model. FIG. 12B shows bioluminescence imaging data in mice treated with CAR-T LCAR-B38M and mice treated with non-transduced T cells (UnT). FIG. 12C shows the study design and bioluminescence images of mice in the CAR-T group and the UnT group. FIG. 12D shows images of livers from UnT-treated mice. FIG. 12E shows an ex vivo luciferase assay validating tumors in the livers of UnT-treated mice.

[0046] FIGS. 13A-13F show clinical parameters of two monkeys treated with LCAR-B38M T-CAR cells. Clinical parameters monitored in the study include body temperature (FIG. 13A), body weight (FIG. 13B), Complete Blood Count (CBC, FIGS. 13C and 13D), as well as serum chemical and cytokine levels (FIGS. . 13E and 13F).

[0047] FIGS. 14A-C show in vitro cytotoxicity assays on LCAR-B38M CAR T-cells and LCAR-B27S CAR T-cells prepared from the same three multiple myeloma patients, respectively. FIG. 14A shows the in vitro cytotoxicity results of LCAR-B38M CAR T-cells and LCAR-B27S CAR T-cells prepared from multiple myeloma patient A. FIG. 14B shows the in vitro cytotoxicity results of LCAR-B38M CAR T-cells and LCAR-B27S CAR T-cells prepared from a patient with multiple myeloma B. FIG. 14C shows the in vitro cytotoxicity results of LCAR-B38M CAR T-cells and LCAR-B27S CAR T-cells prepared from C multiple myeloma patients.

[0048] FIG. 15A compares the structures of a VHH-based CAR and a conventional scFv-based CAR. The schematic structure on the left shows an exemplary monovalent monospecific CAR that has an extracellular antigen-binding domain comprising a VHH domain. The schematic structure on the right shows an exemplary monovalent monospecific CAR that has an extracellular antigen-binding domain comprising a scFv domain.

[0049] FIG. 15B compares the structures of a VHH-based CAR having two antigen-binding sites and a conventional scFv-based CAR having two antigen-binding sites. The schematic structure on the left is an exemplary CAR that has an extracellular antigen-binding domain comprising two VHH domains. The two VHH domains can be the same or different. The schematic structure on the right shows an exemplary CAR having an extracellular antigen-binding domain comprising two scFv domains. The two scFv domains can be the same or different.

[0050] FIG. 15C shows exemplary schematic structures of VHH-based bivalent and bispecific CARs. The schematic structure in the upper left panel shows an exemplary monoepitope, bivalent CAR having an extracellular antigen-binding domain comprising two identical VHH domains, each of which specifically binds epitope 1 of antigen A. The schematic structure in the upper right panel shows an exemplary monoepitope bivalent CAR that has an extracellular antigen-binding domain comprising a first VHH domain specifically binding to epitope 1 of antigen A, and a second VHH domain specifically binding to epitope 2 of antigen A. Epitope 1 and the epitope 2 of antigen A may be different in their structures and/or sequences. The schematic structure in the lower left panel shows an exemplary bispecific CAR having an extracellular antigen-binding domain comprising a first VHH domain specifically binding antigen A, and a second VHH domain specifically binding antigen B. Antigen A and antigen B are different antigens.

[0051] FIG. 15D shows schematic structures of exemplary VHH-based CARs with three or more VHH domains. CARs can have a plurality of VHH domains fused to each other directly or through peptide linkers. VHH domains can be the same or different. Different VHH domains can specifically bind to different epitopes on the same or different antigens.

[0052] FIG. 15E shows exemplary modified immune effector cells that coexpress two different VHH-based CARs. The exemplary modified immune effector cell in the left panel coexpresses two different monospecific and monovalent CARs. The immune effector cell engineered in the central panel coexpresses a monospecific, monovalent CAR and a bispecific or bivalent CAR. The exemplary engineered immune effector cell in the right panel coexpresses two different bispherical or bivalent CARs. CARs can recognize different antigens.

DETAILED DESCRIPTION OF THIS APPLICATION

[0053] The present application provides single-domain anti-BCMA antibodies (sdAb) and chimeric antigen receptors (CARs) comprising an extracellular antigen-binding domain comprising one or more BCMA-binding moieties (such as anti-BCMA sdAbs) . Multivalent CARs comprising at least two binding moieties (such as sdAbs) that specifically bind to a single antigen are also provided. In some embodiments, the present application provides multivalent CARs (such as bivalent or trivalent) comprising at least two anti-BCMA sdAbs. In some embodiments, the at least two anti-BCMA sdAbs are different anti-BCMA sdAbs that specifically bind to different epitopes on BCMA. The anti-BCMA sdAbs, CARs and engineered immune cells expressing CARs described in the present application are useful agents for the treatment of cancer.

[0054] Notably, the present application demonstrated superior efficacy of bivalent biepitope CARs comprising two anti-BCMA sdAbs targeting different BCMA epitopes (e.g., LCAR-B38M), in the treatment of multiple myeloma in human patients. In an interim analysis of a Phase I/II clinical trial, 100% of patients with relapsed or refractory multiple myeloma responded to LCAR-B38M CAR-T treatment. 94% of patients had clear clinical remission of myeloma within two months of receiving CAR-T treatment. Patients who met stringent complete response (SCR) criteria remained free of minimal residual disease after more than a year of CAR-T treatment. Furthermore, LCAR-B38M CAR-T treatment was well tolerated by patients, as most patients only experienced mild and manageable cytokine release syndrome, a common side effect of CAR T cell-based therapy. No patient experienced neurological side effects. In comparison, a pilot clinical study of a monovalent CAR containing a single anti-BCMA sdAb showed a lower objective response rate and complete remission rate, and a higher relapse rate among treated patients. Prior to this application, all BCMA CARs in clinical studies had only a BCMA-binding moiety in the extracellular antigen-binding domain. The improved clinical efficacy and safety of the multivalent BCMA CARs of the present invention are unexpected.

[0055] Accordingly, one aspect of the present application provides a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a plurality of a single-domain antibody (sdAb) that specifically binds to BCMA; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0056] In another aspect, there is provided a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) that specifically binds to a first epitope of BCMA, and a second BCMA-binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope is different from the second epitope.

[0057] New anti-BCMA sdAbs and CARs comprising any one or more of the anti-BCMA sdAbs described herein are also provided.

[0058] Engineered immune effector cells (such as T cells) comprising CARs, pharmaceutical compositions, kits, articles of manufacture and methods of treating cancer using engineered immune effector cells or sdAbs are also described herein.

I. Definitions

[0059] The term "antibody" includes monoclonal antibodies (including full-length 4-chain antibodies or full-length heavy chain antibodies that have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Antibodies contemplated herein include single domain antibodies, such as heavy chain only antibodies.

[0060] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical L) light chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetrameric units along with an additional polypeptide called the J chain and contains 10 antigen-binding sites, while IgA antibodies comprise of 2-5 of the 4-chain basic units that can polymerize to form polyvalent assemblies in combination with the J chain. In the case of IgGs, the 4-chain unit is usually about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the isotype of H chain. Each H and L chain also have disulfide bonds regularly spaced intrachain. Each H chain has at the N-terminus a variable domain (VH) followed by three constant domains (CH) for each of the α and Y chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of the 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, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, Y and μ, respectively. The Y and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0061] The term “heavy chain antibody” or “HCAb” refers to a functional antibody, which comprises heavy chains, but lacks the light chains generally found in 4-chain antibodies. Camelid animals (such as camels, llamas or alpacas) are known to produce HCAbs.

[0062] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are designed from camelid HCAbs, and their heavy chain variable domains are referred to herein as “VHHs”. Some VHHs can also be known as nanobodies. The camelid sdAb is one of the smallest known antibody fragments that bind antigen (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)). A basic VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to structural regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0063] An “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from association with all other components of its production environment. Contaminating components of its production environment, such as those that result from recombinant transfected cells, are materials that would typically interfere with research, diagnosis, or therapeutic uses for the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a rotating cup sequencer, or (3) homogeneity by SDS-PAGE under non-reducing or reducing conditions using blue Coomassie or, preferably, silver coloring. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, the isolated polypeptide or antibody will be prepared by at least one purification step.

[0064] The “variable region” or “variable domain” refers to the amino-terminal domains of the heavy or light chain of the antibody. The heavy chain and light chain variable domains can be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies in the same class) and contain the antigen-binding sites. Only heavy chain antibodies from the Camelid species have a single heavy chain variable region, which is referred to as “VHH”. VHH is thus a special type of VH.

[0065] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not uniformly distributed across the entire range of variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The most highly conserved portions of variable domains are called framework regions (FR). The variable domains of the native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form connecting loops and, in some cases, part of the beta-sheet structure. The HVRs on each chain are held together in close proximity by the FR regions and, with the HVRs on the other chain, contribute to antibody-antibody site formation (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in binding an antibody to an antigen, but have several effector functions, such as the antibody's participation in antibody-dependent cellular toxicity.

[0066] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical, except for possibly naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in smaller amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous as they are synthesized by hybridoma culture, or recombinantly, not contaminated by other immunoglobulins. monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including, for example, 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, 2nd ed. 1988); , in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N. Y., 1981)), recombinant DNA methods (see, e.g., US Patent 4,816,567), phage display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); ):299-310 (2004); 12472 (2004); and Lee et al., J. Immunol. 284(1-2):119-132 (2004), and technologies for producing human or human-like antibodies in animals that possess parts or all of the loci of human immunoglobulin or genes encoding human immunoglobulin sequences (see, for example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Academic. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); US Patent 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0067] The term “naked antibody” refers to an antibody that is not conjugated to a cytotoxic moiety or radioactive label.

[0068] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length 4-chain antibodies include those with heavy and light chains including an Fc region. Full-length heavy chain antibodies include the heavy chain (such as VHH) and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0069] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see US Patent 5,641,870, Example 2; Zapata et al. Protein Eng. 8 (10):1057-1062 [1995]); single-chain antibody molecules; single domain antibodies (such as VHH), and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation that reflects the ability to readily crystallize. The Fab fragment consists of an entire L chain, together with the variable region domain of the H chain (VH), and the first constant domain of a heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activity and is still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments in having some additional residues at the carboxy terminus of the CH1 domain including one or more cysteines of the antibody hinge region. Fab'-SH is the designation here for Fab' in which the cysteine residue(s) of the constant domains have a free thiol group. F(ab‘)2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0070] The Fc fragment comprises the carboxy-terminal portions of the two H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region that is also recognized by Fc receptors (FcR) found on certain cell types.

[0071] "Fv" is the minimal antibody fragment which contains a complete antigen binding and recognition site. This fragment consists of a dimer of a light chain and heavy chain variable region domain in close, non-covalent association. From the folding of these two domains, six hypervariable loops emanate (3 loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

[0072] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the antibody VH and VL domains linked to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VHe VLo domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0073] The "functional fragments" of the antibodies described herein comprise a portion of an intact antibody, generally including the antigen-binding or variable region of the intact antibody or the Fc region of an antibody that retains or has modified FcR binding capacity . Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0074] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the VH and VL domains so that the Inter-chain but not intra-chain pairing of the V domains is obtained, thus resulting in a bivalent fragment, i. e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Natl. Academic. Sci. USA 90:6444-6448 (1993).

[0075] Monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobins) in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a class or particular antibody subclass, while the remainder of the chain(s) is(are) identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, as well as fragments of such antibodies , as long as they exhibit the desired biological activity (U.S. Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest here include PRIMATTZFD® antibodies in which the antigen-binding region of the antibody is derived from an antibody produced by, for example, immunizing monkeys with an antigen of interest. As used herein, "humanized antibody" is a subset of "chimeric antibodies."

[0076] “Humanized” forms of non-human antibodies (e.g., camelids) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues of an HVR (defined below) of the recipient are replaced by residues of an HVR of a non-human species (donor antibody) such as mouse, rat , rabbit or non-human primate with the desired specificity, affinity and/or capacity. In some cases, framework residues ("FR") of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications can be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and in the L chain no more than 3. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more 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, 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 US Patent Nos. 6,982,321 and 7,087,409.

[0077] A “human antibody” is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and/or made using any of the techniques for producing human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising 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). Also available for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opinion. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been engineered to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been inactivated, e.g., immunized xenomice (see, e.g., US Patent 6,075,181 and 6,150,584 in relation to XENOMOUSE™ technology). See also, for example, Li et al. Proc. Natl. Academic. Sci. USA, 103: 3557-3562 (2006) on human antibodies generated through a human B cell hybridoma technology.

[0078] The term "hypervariable region", "HVR" or "HV", when used herein, refers to regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops. Generally, sdAbs comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 exhibits the greatest diversity of the three HVRs, and is believed to play a unique role in imparting fine specificity to antibodies. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0079] The term "Complementarity Determination Region" or "CDR" is used to refer to hypervariable regions as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)

[0080] A number of HVR delimitations are in use and are covered here. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on an analysis of available complex crystal structures. The results of each of these HVRs are noted in Table 1. Table 1. HVR delimitations

[0081] HVRs may include "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL and 26 -35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0082] The amino acid residues of an sdAb (such as VHH) are numbered according to the general numbering for VH domains provided by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to the VHH domains of Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2):185-195. According to this numbering, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. In this regard, it should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated. by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering).

[0083] The expression "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refers to the numbering system used for heavy chain variable domains or heavy chain variable domains. light chain of antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain more or fewer amino acids corresponding to a shortening or insertion into a variable domain FR or HVR. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and the inserted residues (e.g. residues 82a, 82b and 82c, etc. according to Kabat) after heavy chain RF residue 82. Kabat residue numbering can be determined for a given antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence.

[0084] Unless otherwise indicated, the numbering of residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The "EU index as in Kabat" refers to the residue numbering of the human UE IgG1 antibody.

[0085] "Framework" or "FR" residues are variable domain residues other than HVR residues as defined herein.

[0086] A “human consensus framework” or “human acceptor framework” is a framework that represents the most common amino acid residues in a selection of VL or VH human immunoglobulin framework sequences. Human VL or VH is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include for the VL, the subgroup may be the kappa I, kappa II, kappa III or kappa IV subgroup as in Kabat et al., supra. Additionally, for VH, the subgroup can be subgroup I, subgroup II or subgroup III Kabat et al. Alternatively, a human consensus framework may be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework, by aligning the donor framework sequence with a collection of various framework sequences human. A human acceptor structure “derived from” a human immunoglobulin structure or a human consensus structure may comprise the same amino acid sequence, or may contain changes to the pre-existing amino acid sequence. In some embodiments, the number of pre-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 .

[0087] An "amino acid modification" at a specified position, for example, of 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. Insertion "adjacent" to a specified residue means insertion within one to two residues thereof. The insertion can be N-terminal or C-terminal to the specified residue. The preferred amino acid modification here is a substitution.

[0088] An "affinity matured" antibody is one with one or more changes in one or more HVRs that result in an improvement in the affinity of the antibody for the antigen, compared to a parent antibody that does not have the change(s). ions). In some embodiments, the affinity-matured antibodies have nanomolar or even picomolar affinities for a target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by VH- and VL domain shuffling. Random mutagenesis of HVR residues and/or framework residues is described by, for example: 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).

[0089] As used herein, the term "specifically binds", "specifically recognizes", or is "specific for", refers to measurable and reproducible interactions, such as binding between a target and a target-binding protein. antigen (such as a CAR or an sdAb), which is determinant of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antigen-binding protein (such as a CAR or an sdAb) that specifically binds to a target (which may be an epitope) is an antigen-binding protein (such as a CAR or an sdAb) that binds that target with greater affinity, avidity, more readily and/or longer duration than it binds other targets. In some embodiments, the extent of binding of an antigen-binding protein (such as a CAR or an sdAb) to an unrelated target is less than about 10% of the binding of the antigen-binding protein (such as a CAR or an sdAb). an sdAb) to the target as measured, for example, by a radioimmunoassay (RIA). In some embodiments, an antigen-binding protein (such as a CAR or an sdAb) that specifically binds to a target has a dissociation constant (Kd) of ! 1 μM, ! 100 nM, ! 10 nM, ! 1 nM or ! 0.1 nM, In some embodiments, an antigen-binding protein (such as a CAR or an sdAb) specifically binds to an epitope on a protein that is conserved among the protein of different species. In some embodiments, specific binding may include, but does not require, exclusive binding.

[0090] The term "specificity" refers to the selective recognition of an antigen-binding protein (such as a CAR or an sdAb) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecific", as used herein, indicates that an antigen-binding protein (such as a CAR or an sdAb) has two or more antigen-binding sites, at least two of which bind different antigens. "Bispecific", as used herein, indicates that an antigen-binding protein (such as a CAR or an sdAb) has two different antigen-binding specificities. The term "monospecific" CAR as used herein indicates an antigen-binding protein (such as a CAR or an sdAb) that has one or more binding sites, each of which binds the same antigen.

[0091] The term "valent", as used herein, indicates the presence of a specified number of binding sites on an antigen-binding protein (such as a CAR or an sdAb). A natural antibody for example or a full-length antibody has two binding sites and is bivalent. As such, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" denote the presence of two binding sites, three binding sites, four binding sites, five binding sites and six binding sites, respectively. , on an antigen-binding protein (such as a CAR or an sdAb).

[0092] "Antibody effector functions" refer to biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; upregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. "Reduced or minimized" antibody effector function means that it is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 96%, 97% 98%, 99%) of wild-type or unmodified antibody. Determination of the effector function of the antibody is easily determinable and measurable by one skilled in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement-dependent cytotoxicity and antibody-dependent cytotoxicity are affected. In some embodiments, the effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., “no effect mutation”. In one aspect, the no effect mutation is an N297A or DANA (D265A + N297A) mutation in the CH2 region, Shields et al., J. Biol. Chem. 276 (9):6591-6604 (2001). Alternatively, additional mutations resulting in reduced or eliminated effector function include: K322A and L234A/L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli) or 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).

[0093] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) allow these cytotoxic effector cells to specifically bind to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. Antibodies "arm" cytotoxic cells and are necessary to kill the target cell by this mechanism. The primary cells to mediate ADCC, NK cells, express FCYRIII only, since monocytes express FCYRI, FCYRII, and FcyRIII. Fc expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay such as described in US Patent 5,500,362 or 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS USA 95:652-656 (1998).

[0094] The term "Fc region" here is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of human IgG heavy chain is typically defined to stretch from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. . The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding an antibody heavy chain. antibody. Accordingly, an intact antibody composition may comprise populations of antibodies with all K447 residues removed, populations of antibodies without K447 residues removed, and populations of antibodies having a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0095] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody or a CAR) and its binding partner (e.g., a antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen, or CAR and antigen). . The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, which can be used for the purposes of the present application. Illustrative modalities and specific examples for measuring binding affinity are described below.

[0096] A "blocking" antibody or an "antagonistic" antibody is one that inhibits or reduces the biological activity of the antigen it binds. In some embodiments, the blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0097] "Percentage (%) amino acid sequence identity" and "homology" to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN software. ™ (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the total length of the sequences being compared.

[0098] The "chimeric antigen receptor" or "CAR", as used herein, refers to genetically modified receptors, which can be used to graft one or more antigen specificities onto immune effector cells, such as T cells. Some CARs are also known as "artificial T cell receptors", "chimeric T cell receptors" or "chimeric immune receptors". In some embodiments, the CAR comprises an extracellular antigen-binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and/or other receptors. “CAR-T” refers to a T cell that expresses a CAR. “BCMA CAR” refers to a CAR with an extracellular binding domain specific for BCMA. "Bi-epitope CAR" refers to a CAR with an extracellular binding domain specific for two different epitopes on BCMA.

[0099] An "isolated" nucleic acid molecule encoding a CAR or an sdAb described herein is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free from association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies present herein are in a form different from the form or configuration in which they are found in nature. Isolated nucleic acid molecules are therefore distinguished from the nucleic acid that encodes the polypeptides and antibodies that exist naturally in cells.

[0100] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0101] The nucleic acid is “operationally linked” which is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a secretory presequence or main sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. In general, “operationally linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not need to be contiguous. Binding is accomplished by binding at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0102] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to here as “expression vectors”.

[0103] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that will later be reintroduced into the individual.

[0104] "Allogenic" refers to a graft derived from a different individual of the same species.

[0105] The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary cell and its progeny.

[0106] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transfectants" and "transfected cells" include the primary cell and the cultures derived therefrom, without considering the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same biological function or activity tested in the originally transformed cell are included.

[0107] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to the host cells into which the exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the transformed primary cell and the progeny resulting therefrom, regardless of the number of passages. The offspring may not be completely identical in nucleic acid content to a parental cell, but may contain mutations. Mutant progeny that have the same biological function or activity as that screened or selected for in the originally transformed cell are included here.

[0108] As used herein, "treatment" or "treating is an approach to obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviate one or more symptoms resulting from the disease, decrease the extent of the disease, stabilize the disease (e.g., preventing or delaying worsening of the disease), prevent or delay the spread (e.g., metastasis) of the disease , prevent or delay the recurrence of the disease, delay or reduce the progression of the disease, improve the state of the disease, provide a remission (partial or complete) of the disease, decrease the dose of one or more other medications necessary to treat the disease, delay the progression of the disease, increase the quality of life and/or prolong survival. Also covered by "treatment" is a reduction in the pathological consequence of the cancer. The methods of the present application contemplate any one or more of these aspects of the treatment.

[0109] As used herein, an "individual" or a "subject" refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent or primate. In some embodiments, the individual is a human.

[0110] The term "effective amount" used herein refers to an amount of an agent, such as an SDAb, an engineered immune effector cell, or a pharmaceutical composition thereof, sufficient to treat a specified disorder, condition, or disease, such as how to improve, alleviate, reduce and/or delay one or more of your symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or slow the rate of tumor growth (such as to suppress tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. The effective amount of the drug or composition can: (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, delay, reduce to some extent, and preferably prevent the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., reduce to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and/or recurrence of the tumor; and/or (vii) alleviate, to some extent, one or more of the symptoms associated with cancer.

[0111] "Adjuvant setting" refers to a clinical setting in which an individual has had a history of cancer, and has generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (e.g. , surgical resection), radiotherapy and chemotherapy. However, due to their history of cancer, these individuals are considered at risk for developing the disease. Treatment or administration in the "adjuvant setting" refers to a subsequent mode of treatment. The degree of risk (for example, whether an individual in the adjuvant setting is considered "high risk" or "low risk") depends on several factors, most generally the degree of the disease when first treated.

[0112] "Neoadjuvant Setting" refers to a clinical setting in which the method is performed prior to primary/definitive therapy.

[0113] As used herein, "delaying" the development of cancer means postponing, hindering, reducing, delaying, stabilizing and/or postponing the development of the disease. This delay may vary over different periods of time, depending on the history of the disease and/or the individual being treated. As is evident to one skilled in the art, a sufficient or significant delay may, in fact, encompass prevention, insofar as the individual does not develop the disease. A method that "slows" the development of cancer is a method that reduces the probability of developing the disease in a given period of time and/or reduces the extent of the disease in a given period of time, when compared to not using the method. Such comparisons are usually based on clinical studies using a statistically significant number of subjects. The development of cancer may be detectable using standard methods including, but not limited to, computerized axial tomography (CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound, coagulation tests, arteriography or biopsy. Development can also refer to the progression of cancer that may be initially undetectable and includes occurrence, recurrence, and onset.

[0114] The term "pharmaceutical formulation" refers to a preparation that is in the form of allowing the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to an individual to whom the formulation would be administered. . Such formulations are sterile. A "sterile" formulation is aseptic or free of all living microorganisms and their spores.

[0115] "Carriers", as used herein, include pharmaceutically acceptable carriers, excipients or stabilizers that are not toxic to the cell or mammal that is exposed to them at the dosages and concentrations used. Often, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m- cresol); low molecular weight polypeptide (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming contractions, such as sodium; metal complexes (e.g. protein-Zn complexes); and/or non-ionic surfactants, such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™ or polyethylene glycol (PEG).

[0116] The "diluent" of interest here is one that is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for preparing a liquid formulation, such as a formulation reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In an alternative embodiment, the diluents may include aqueous solutions of salts and/or buffers.

[0117] A "preservative" is a compound that can be added to formulations here to reduce bacterial activity. The addition of a preservative can, for example, facilitate the production of a multidose (multiple dose) formulation. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethylene chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative here is benzyl alcohol.

[0118] A "stable" formulation is one in which the protein in it essentially maintains its physical and chemical stability and integrity after storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N. Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature over a selected period of time. For rapid screening, the formulation can be kept at 40°C for 2 weeks to 1 month, at which time stability is measured. When the formulation is to be stored at 2-8°C, generally the formulation should be stable at 30°C or 40°C for at least 1 month and/or stable at 2-8°C for at least 2 years. When the formulation must be stored at 30°C, generally the formulation must be stable for at least 2 years at 30°C and/or stable at 40°C for at least 6 months. For example, the extent 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% and preferably less than about 5% of the protein is present as an aggregate in the formulation. In other embodiments, any increase in aggregate formation during storage of the formulation can be determined.

[0119] A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed throughout. The reconstituted formulation is suitable for administration (e.g., subcutaneous administration) to a patient being treated with the protein of interest and, in some embodiments of the present application, may be one that is suitable for parenteral or intravenous administration.

[0120] An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure of about 250 to 350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure below that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice freezing osmometer, for example. The formulations of the present invention are hypertonic as a result of the addition of salt and/or buffer.

[0121] It is understood that the embodiments of the present application described herein include "consisting of" and/or "consisting essentially of" embodiments.

[0122] Reference to "about" a value or parameter here includes (and describes) variations of which are directed to that value or parameter per se. For example, the description that refers to "about X" includes the description of "X".

[0123] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means that the method is used to treat cancer of types other than X.

[0124] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0125] As used herein and in the appended claims, the singular forms "a", "or" and "the" include the referring plurals, unless the context clearly specifies otherwise.

II. Anti-BCMA Single Domain Antibodies

[0126] One aspect of the present application provides isolated single-domain antibodies (referred to herein as "anti-BCMA sdAbs") that specifically bind to BCMA, such as human BCMA. In some embodiments, the anti-BCMA sdAb modulates BCMA activity. In some embodiments, the anti-BCMA sdAb is an antagonist antibody. Additionally, antigen-binding fragments derived from any of the anti-BCMA sdAbs described herein, and antigen-binding proteins comprising any of the anti-BCMA sdAbs described herein are provided. Examples of anti-BCMA sdAbs are listed in Table 2 below. Table 2. Exemplary anti-BCMA SdAbs.

[0127] B cell mature 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 exclusively 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 B cell activating factor (BAFF) and a proliferation including ligand (APRIL) (e.g., Mackay et al., 2003 and Kalled et al., Immunological Review, 204:43-54, 2005) . BCMA may be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma. High affinity antibodies can block the binding between BCMA and its native ligands BAFF and APRIL. Anti-BCMA sdAbs can be used in combination with cellular immunotherapy using CAR T-cells, for example, to enhance cytotoxic effects against tumor cells.

[0128] In some embodiments, an anti-BCMA sdAb comprising one, two or all three CDRs of the amino acid sequence of SEQ ID NO:115 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:116 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:117 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:118 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:119 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:120 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:121 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:122 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:123 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:124 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:125 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:126 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:127 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:128 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:129 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:130 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:131 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:132 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:133 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:134 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:135 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:136 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:137 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:138 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:139 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:140 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:141 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:142 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:143 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:144 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:145 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:146 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:147 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:148 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:149 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:150 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:151 is provided. In some embodiments, an anti-BCMA sdAb comprising one, two, or all three CDRs of the amino acid sequence of SEQ ID NO:152 is provided. 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 acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.

[0129] In some embodiments, there is provided an anti-BCMA sdAb comprising at least one, at least two or all three CDRs selected from (a) a CDR1 comprising an amino acid sequence selected from SEQ ID NO: 1-38; (b) a CDR2 comprising an amino acid sequence selected from SEQ ID NO:39-76; and (c) a CDR3 comprising an amino acid sequence selected from SEQ ID NO: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 acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.

[0130] In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 with at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 138; (b) a CDR2 of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 39-76; and (c) a CDR3 of at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NO:77-114. In some embodiments, a CDR having at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-BCMA sdAb comprising that sequence retains the ability to bind to BCMA. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 with about any of 1, 2, 3, or 4 amino acid substitutions (e.g., conservative substitutions), insertions or deletions to a amino acid sequence selected from SEQ ID NO: 1-38; (b) a CDR2 with about any of 1, 2, 3 or 4 amino acid substitutions (e.g., conservative substitutions), insertions or deletions to an amino acid sequence selected from SEQ ID NO: 39-76; and (c) a CDR3 with about any of 1, 2, 3 or 4 amino acid substitutions (e.g., conservative substitutions), insertions or deletions to an amino acid sequence selected from SEQ ID NO:77-114. In some embodiments, the anti-BCMA sdAb is affinity matured. In some embodiments, the anti-BCMA sdAb is camelid. In some embodiments, the anti-BCMA sdAb is humanized. In some embodiments, the anti-BCMA sdAb comprises a human acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.

[0131] In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:77. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:78. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:79. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:80. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:81. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:82. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:83. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:84. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:85. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:86. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:87. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:88. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:89. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:52; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:90. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:91. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:92. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:93. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:94. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:96. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:98. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:99. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:100. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:101. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:102. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:103. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:104. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:105. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:106. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:107. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:108. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:109. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:110. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:111. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:112. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:113. In some embodiments, there is provided an anti-BCMA sdAb comprising three CDRs comprising: (a) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; (b) a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and (c) a CDR3 comprising the amino acid sequence of SEQ ID NO:114. In some embodiments, the anti-BCMA sdAb is camelid. In some embodiments, the anti-BCMA sdAb is humanized. In some embodiments, the anti-BCMA sdAb comprises a human acceptor structure, for example, a human immunoglobulin structure or a human consensus structure.

[0132] In some embodiments, the anti-BCMA sdAb, including any of the modalities described above (i.e., anti-BCMA sdAb comprising specific CDR1, CDR2 and/or CDR3 comprises a VHH domain with at least about 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence of amino acids selected from SEQ ID NO:115-152, in some embodiments, a VHH sequence with at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-BCMA sdAb comprising this sequence maintains the ability to bind BCMA. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted/or deleted in an amino acid sequence selected from SEQ ID NO: 115-152. insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-BCMA sdAb comprises an amino acid sequence selected from SEQ ID NO:115-152, including post-translational modifications of that sequence.

[0133] In some embodiments, there is provided an isolated anti-BCMA sdAb comprising a VHH domain with an amino acid sequence selected from the group consisting of SEQ ID NOs:115-152. In some embodiments, there is provided a multivalent polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:115-152.

[0134] In some embodiments, functional epitopes can be mapped by combined alanine scanning. In this process, a combinatorial alanine scanning strategy can be used to identify amino acids in the BCMA protein that are required for interaction with anti-BCMA sdAbs. In some embodiments, the epitope is conformational and the crystal structure of anti-BCMA sdAbs bound to BCMA can be employed to identify the epitopes. In some embodiments, the present application provides a BCMA epitope derived from an amino acid sequence selected from the group consisting of SEQ ID NOs:388-394. In some embodiments, the present application provides a BCMA epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:388-394.

[0135] In some embodiments, the present application provides antibodies that compete with any of the anti-BCMA sdAbs described herein for binding to BCMA. In some embodiments, the invention provides antibodies that compete with the anti-BCMA sdAbs provided herein for binding to an epitope on BCMA. In some embodiments, there is provided an antibody 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 BCMA competitively with an anti-BCMA sdAb comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:115-152.

[0136] In some embodiments, competition assays can be used to identify a monoclonal antibody that competes with an anti-BCMA sdAb described here for binding to BCMA. Competition assays can be used to determine whether two antibodies bind to the same epitope, recognizing identical or sterically overlapping epitopes, or whether one antibody competitively inhibits the binding of another antibody to the antigen. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a BCMA epitope derived from an amino acid sequence selected from the group consisting of SEQ ID NOs:388-394) that is bound to an antibody described herein. Exemplary competition assays include, but are not limited to, routine assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In some embodiments, two antibodies are said to bind to the same epitope if each blocks the binding of the other by 50% or more. In some embodiments, the antibody that competes with an anti-BCMA sdAb described herein is a camelid, chimeric, humanized, or human antibody. In some embodiments, the present application provides an antibody that competes with a camelid, chimeric, humanized or human anti-BCMA sdAb, as described herein.

[0137] In some embodiments, an anti-BCMA antibody or antigen-binding protein comprising any of the anti-BCMA sdAbs described above is provided. In some embodiments, the anti-BCMA antibody is a monoclonal antibody, including a camelid, chimeric, humanized, or human antibody. In some embodiments, the anti-BCMA antibody is an antibody fragment, e.g., a VHH fragment. In some embodiments, the anti-BCMA antibody is a full-length heavy chain antibody that comprises 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.

[0138] In some embodiments, the anti-BCMA antibody (such as the anti-BCMA sdAb) or antigen-binding protein according to any of the previous embodiments can incorporate any of the characteristics, alone or in combination, as described in Secs. 1-7 of “Characteristics of antibodies” below.

[0139] In some embodiments, an isolated nucleic acid encoding any 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 wherein the nucleic acid comprises a sequence having at least about any of 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity for a nucleic acid sequence selected from the group consisting of SEQ ID NO :153-190. In some embodiments, an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:153-190 is provided. In some embodiments, a vector (e.g., expression vector) comprising such nucleic acid. In some embodiments, a host cell comprising such nucleic acid is provided. In some embodiments, a method of producing an anti-BCMA antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-BCMA antibody, as provided above, under conditions suitable for expression of the antibody. anti-BCMA, and optionally recovering the anti-BCMA antibody from the host cell (or host cell culture medium).

Characteristics of antibodies 1. Antibody affinity

[0140] In some embodiments, an anti-BCMA antibody provided herein has a dissociation constant (Kd) of < 1μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10-8 M or less e.g. 10-8 M to 10-13 M e.g. 10-9 M to 10-13 M).

[0141] In some embodiments, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version or VHH fragment of an antibody of interest and its antigen as described by the following assay. For example, the binding affinity of Fab solution for antigen is measured by equilibrating Fab with a minimum concentration of labeled antigen (125I) in the presence of a titration series of unlabeled antigen, then capturing bound antigen with a plate. coated with anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293:865-881(1999)).

[0142] In some embodiments, Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with CM5 antigen chips. immobilized at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of the Supplier. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (~0.2 μM) prior to injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RH ) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two serial dilutions of Fab or VHH of the antibody of interest (0.78 nM to 500 nM) are injected into PBS with 0.05% polysorbate 20 surfactant (TWEEN-20TM) (PBST) at 25° C at a flow rate of approximately 25 μl/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE ® Assessment Software version 3.2) by simultaneously fitting the association and dissociation models. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate passes 106 M-1 s-1 by the surface plasmon resonance assay above, then the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in the emission intensity of fluorescence (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25oC from a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, such as measured on a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or an SLM-AMINCO TM 8000-series spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

[0143] In some embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv, VHH, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat Med 9:129-134 (2003). For a review of scFv fragments, see for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458). For discussion of Fab and F(ab')2 fragments comprising salvage receptor-binding epitope residues and having an increase in half-life in vivo, see US Patent 5,869,046.

[0144] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Academic. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0145] Antibody fragments can be produced by various techniques, including, but not limited to, proteolytic digestion of an intact antibody, as well as production by recombinant host cells (e.g. E. coli or phage), as described herein .

3. Chimeric and humanized antibodies

[0146] In some embodiments, the antibody provided here is a chimeric antibody. Certain chimeric antibodies are described, for example, in US Patent 4,816,567; and Morrison et al., Proc. Natl. Academic. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a camelid species such as llama) and a human constant region. In another example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from the parent antibody. Chimeric antibodies include antigen-binding fragments.

[0147] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues of a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve specificity or the affinity of the antibody.

[0148] Humanized antibodies and methods of preparing them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR graft (a-CDR)); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurface”); Dall’Acqua et al., Methods 36:43–60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0149] Human structural regions that can be used for humanization include, but are not limited to: framework regions selected using the “best fit” method (see, for example, Sims et al. J. Immunol. 151:2296 (1993)); structural regions derived from the human antibody consensus sequence of a particular subgroup of light or heavy chain variable regions (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); mature (somatically mutated) human framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and structural regions derived from screening of FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611 -22618 (1996)).

[0150] In some embodiments, the SdAbs are modified, such as humanized, without decreasing the native affinity of the domain for the antigen and at the same time reducing its immunogenicity relative to a heterologous species. For example, the amino acid residues of the antibody variable domain (VHH) of a llama antibody can be determined, and one or more of the camelid amino acids, for example, in the framework regions, are replaced by their human counterpart, as found. in the human consensus sequence, without this polypeptide having lost its typical character, i. It is. humanization does not significantly affect the antigen-binding capacity of the resulting polypeptide. Humanization of camelid sdAbs requires the introduction and mutagenesis of a limited amount of amino acids into 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, the light and heavy chain, and the preservation of the assembly of both chains.

[0151] Single-domain antibodies comprising a VHH domain can be humanized to have human-like sequences. In some embodiments, the FR regions of the VHH domain used herein comprise at least about 50%, 60%, 70%, 80%, 90%, 95% or more amino acid sequence homology to human VH framework regions. . An exemplary class of humanized VHH domains is characterized by the fact that VHHs carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45 , such as, for example, L45 and a tryptophan in position 103, according to Kabat's numbering. As such, polypeptides belonging to this class show high amino acid sequence homology to human VH framework regions and said polypeptides can be administered to a human directly without the expectation of an undesired immune response and without the burden of additional humanization.

[0152] Another exemplary class of humanized camelid sdAbs was described in WO 03/035694 and contains hydrophobic FR2 residues typically found in conventional antibodies of human origin or other species, but compensating for this loss in hydrophilicity by the arginine residue charged at the position 103 that replaces the conserved tryptophan residue present in VH of single-chain antibodies. As such, peptides belonging to these two classes show high amino acid sequence homology to human VH framework regions and said peptides can be administered to a human directly without the expectation of an unwanted immune response and without the burden of additional humanization.

4. Human Antibodies

[0153] In some embodiments, an antibody provided herein is a human antibody. 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. Opinion. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opinion. 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 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0154] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci were generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, Patent. US 6,075,181 and 6,150,584 describing XENOMOUSETM technology; Patent. U.S. 5,770,429 describing HUMAB® technology; US Patent 7,041,870 describing K-M MOUSE® technology, and US Patent Application Publication 2007/0061900, describing VELOCIMOUSE® technology). The human variable regions of intact antibodies generated by such animals can be further modified, for example, by combining with a different human constant region.

[0155] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Academic. Sci USA, 103:3557-3562 (2006). Additional methods include those described, for example, in US Patent 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (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).

[0156] Human antibodies can also be generated by isolating variable domain sequences from Fv clones selected from phage display libraries of human origin. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0157] One technique for obtaining VHH sequences directed against a particular antigen or target involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e., so as to raise an immune response and/or heavy chain antibodies directed against said antigen or target), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said VHH sequences (such as a blood sample, serum sample or B cell sample) and, in then generating VHH sequences directed against said antigen or target, from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, mice expressing heavy chain antibody and the additional methods and techniques described in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens et al., Proc. Natl. Academic. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can be used. For example, such heavy chain antibody-expressing mice can express heavy chain antibodies with any suitable (unique) variable domain, such as (unique) variable domains from natural sources (e.g., human (unique) variable domains, camelid variable domains (unique) or shark (unique) variable domains, as well as, for example, synthetic or semi-synthetic (unique) variable domains.

5. Library-derived antibodies

[0158] The antibodies of the present application can be isolated by screening combinatorial libraries of antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies that have the desired binding characteristics. These methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in the 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. Academic. 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 have been described, for example, see US Patent 7,371,849.

[0159] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically displays antibody fragments, either as single-chain Fv (scFv) fragments or Fab fragments. Immunized source libraries provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the naive repertoire can be cloned (e.g. from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12 :725-734 (1993). Finally, naïve libraries can also be produced synthetically by cloning non-rearranged V gene segments from stem cells, and using PCR primers containing a random sequence to encode the highly variable CDR3 regions and to achieve in vitro rearrangement. as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent 5,750,373, and US Patent Publication 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/ 0237764, 2007/0292936, and 2009/0002360.

[0160] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments here.

6. Multispecific antibodies

[0161] In some embodiments, an antibody provided herein is a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are antibodies that have binding specificities for at least two different sites. In some embodiments, one of the binding specificities is for an antigen selected from the group consisting of CD19, CD20, BCMA, and CD38, and the other is for any other antigen. In some embodiments, the bispecific antibodies can bind to two different epitopes of an antigen selected from the group consisting of CD19, CD20, BCMA and CD38. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing an antigen selected from the group consisting of CD19, CD20, BCMA, and CD38.

[0162] Bispecific antibodies can be prepared as complete antibodies or antibody fragments. Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10:3655 (1991)), “knob-in-hole” engineering (see, for example, US Patent 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic direction effects to produce heterodimeric antibody Fc molecules (WO 2009/089004A1); crosslinking two or more antibodies or fragments (see, for example, US Patent 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using “diabody” technology to make bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991); and creating polypeptides comprising tandem single-domain antibodies (see, for example, Patent Application 20110028695; and Conrath et al. J. Biol. Chem., 2001; 276 (10):7346-50). Engineered antibodies with three or more functional antigen-binding sites, including “Octopus Antibodies”, are also included herein (see, for example, US 2006/0025576A1).

7. Antibody Variants

[0163] In some embodiments, amino acid sequence variants of the antibodies provided here are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody 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 of, and/or insertions and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, as long as the final construct has the desired characteristics, for example, antigen binding.

a) substitution, insertion and deletion variants

[0164] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 3 under the heading “Preferred Substitutions”. More substantial changes are provided in Table 3, under the heading of “exemplary substitutions”, and as further described below with reference to the acidic amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/enhanced antigen binding, reduced immunogenicity, or improved ADCC or CDC. TABLE 3. Amino Acid Substitutions

[0165] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acids:Asp, Glu; (4) basic:His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatics:Trp, Tyr, Phe.

[0166] Non-conservative substitutions will imply exchanging a member of one of these classes for another class.

[0167] One type of substitution variant involves replacing one or more residues of the hypervariable region of a parental antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the antibody. parental. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0168] Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made at HVR “hot spots,” that is, residues encoded by codons that mutate at high frequency during the process of somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179- 196 (2008)), and/or SDRs (a-CDRs), with the resulting VH or VL variant being tested for binding affinity. Affinity maturation through construction and reselection of secondary libraries has been 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 forms of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., by error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves HVR-targeted approaches, in which multiple 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 or templating mutagenesis. CDR-H3 and CDR-L3, in particular, are often targeted.

[0169] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs. These changes may be outside of the "key points" of the HVR or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR is unchanged, or contains no more than one, two, or three amino acid substitutions.

[0170] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a target residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid locations that demonstrate functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify points of contact between the antibody and antigen. These contact residues and neighboring residues can be targeted or eliminated as candidates for replacement. Variants can be screened to determine whether they contain the desired properties.

[0171] Amino acid sequence insertions include amino and/or carboxyl terminal fusions varying in length ranging from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusion at the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

b) Glycosylation variants

[0172] In some embodiments, an antibody provided herein is altered 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 accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0173] When the antibody comprises an Fc region, the carbohydrate linked to them can be changed. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally linked by an N-bond to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, for example, mannose, N-acetyl-glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose linked to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the present application can be made in order to create antibody variants with certain improved properties.

[0174] In some embodiments, antibody variants are provided with a carbohydrate backbone that is devoid of fucose linked (directly or indirectly) to an Fc region. For example, the amount of fucose of such an antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures linked to Asn 297 (e.g., complex, hybrid, and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i. e., between positions 294 and 300, due to minor sequence variations in the antibodies. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; 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 CHO Lecl3 cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application 2003/0157108 A1, Presta, L ; and WO 2004/056312 A1, Adams et al., especially in Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, CHO cells knockout (see, for example, Yamane -Ohnuki et al. Bioeng. 87:614 (2004);

[0175] Antibody variants are further provided with bisected oligosaccharides, for example, in which a biantennary oligosaccharide linked 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 WO 2003/011878 (Jean-Mairet et al.); US Patent 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc region variants

[0176] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating a variant of the Fc region. The Fc region variant may comprise a human Fc region sequence (e.g., an IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0177] In some embodiments, the present application contemplates an antibody variant that has some, but not all, effector functions, which makes it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody is devoid of binding to FcyR (hence probably no ADCC activity) but retains the ability to bind to FcRn. The primary cells for mediating ADCC, NK cells, express Fc(RIII only while monocytes express Fc(RI, Fc(RII, and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in US Patent 5,500,362 (see, for example Hellstrom, I. et al. Proc. Nat'l Acad. USA 83:70597063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. ; 5, 821, 337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® nonradioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include blood mononuclear cells peripheral (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and is therefore devoid of CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045 -1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). Determinations of FcRn binding and in vivo clearance/half-life can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18 (12):1759- 1769 (2006)).

[0178] Antibodies with reduced effector function include those with substitution of one or more of the Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Patent 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. 7,332,581).

[0179] Certain antibody variants with improved or decreased binding to FcRs are described. (See, for example, US Patent 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0180] In some embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and/or 334 of the Fc region (EU residue numbering) .

[0181] In some embodiments, changes are made to the Fc region that result in altered (i.e., improved or decreased) C1q binding and/or complement-dependent cytotoxicity (CDC), for example, as described in US Patent 6,194. 551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0182] Antibodies with increased half-lives and better binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al. al. J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Such antibodies comprise an Fc region with one or more substitutions within it that improve binding of the Fc region to FcRn. Such Fc variants include those with 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, replacement of the Fc434 region residue (US Patent 7,371,826).

[0183] See also Duncan & Winter, Nature 322:738-40 (1988); US Patent 5,648,260; US Patent 5,624,821; and WO 94/29351 relating to other examples of Fc region variants. d) Cysteine-engineered antibody variants

[0184] In some embodiments, it may be desirable to create cysteine-engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are replaced with cysteine residues. In particular embodiments, the substituted residues occur in accessible sites on the antibody. By replacing the residues with cysteine, reactive thiol groups are thereby positioned at accessible sites on the antibody and can be used to conjugate the antibody to other molecular moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, such as described here further. In some embodiments, any one or more of the following residues may be substituted for cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the Fc region of the heavy chain. Cysteine-modified antibodies can be generated as described, for example, in US Patent 7,521,541. e) Antibody Derivatives

[0185] In some embodiments, an antibody provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable fractions 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 copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols ( e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers bound to the antibody can vary, and if more than one polymer is bound, these may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0186] In some embodiments, conjugates of an antibody and non-proteinaceous fraction that can be selectively heated by exposure to radiation are provided. In some embodiments, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm normal cells but that heat the non-proteinaceous fraction to a temperature at which cells proximal to the non-proteinaceous antibody fraction are dead. Preparation methods

[0187] The antibodies (such as sdAbs) described herein can be prepared using any method known in the art or as described herein.

[0188] Methods for preparing sdAbs have been described. See, for example, Els Pardon et al, Nature Protocol, 2014; 9(3):599. Single domain antibodies (such as VHHs) can be obtained using methods known in the art such as by immunizing a Camelid species (such as camel or llama) and obtaining hybridomas from it, or by cloning a library of sdAbs using molecular biology known in the art and subsequent selection by ELISA with individual clones from unselected libraries or using phage display.

[0189] For the recombinant production of sdAbs, the nucleic acid encoding the sdAbs can be isolated and inserted into a replicable vector for further cloning (DNA amplification) or for expression. The DNA encoding the sdAb is easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the antibody's heavy and light chains). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, the preferred host cells are of prokaryotic or eukaryotic (usually mammalian) origin.

1. Polyclonal Antibodies

[0190] Polyclonal antibodies are generally created in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to an immunogenic protein in the species to be immunized, e.g. keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g. of maleimidobenzoyl sulfosuccinimide (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are independently lower alkyl groups. Examples of adjuvants that can be used include complete Freund's adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol can be selected by one skilled in the art without undue experimentation.

[0191] Animals are immunized against the antigen, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg or the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally in multiple sites. One month later, animals are boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the serum is tested for antibody titer. The animals are reinforced until the title is equal. Conjugates can also be prepared in recombinant cell culture as protein fusions. Furthermore, aggregating agents such as alum are suitable for enhancing the immune response.

2. Monoclonal Antibodies

[0192] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical, except for possibly naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in smaller quantities. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0193] For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al. Nature, 256:495 (1975), or can be prepared by recombinant DNA methods (US Patent 4,816,567).

[0194] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described herein above to trigger lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).

[0195] The immunizing agent will typically include the antigenic protein or a fusion variant thereof. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies:Principles and Practice, Academic Press (1986), pp. 59-103.

[0196] Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are seeded and cultivated in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells do not possess the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT deficient cells.

[0197] Preferred immortalized myeloma cells are those that fuse efficiently, sustain stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, murine myeloma lines, such as those derived from mouse tumors MOPC-21 and MPC-11 available at the Salk Institute Cell Distribution Center, San Diego, California, are preferred. USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Va. USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0198] The culture medium in which the hybridoma cells are grown undergoes assays for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0199] The culture medium in which the hybridoma cells are grown can be assayed for the presence of monoclonal antibodies directed against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al. Anal. Biochem. 107:220 (1980).

[0200] After hybridoma cells that produce antibodies of desired specificity, affinity and/or activity are identified, clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, hybridoma cells can be cultured in vivo as tumors in a mammal.

[0201] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascitic fluid or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or chromatography affinity.

[0202] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent 4,816,567, and as described above. DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulin protein, so as to synthesize monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of antibody-encoding DNA include Skerra et al., Curr. Opinion in Immunol., 5:256262 (1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

[0203] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using libraries of phages. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain "shuffling" (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0204] DNA can also be modified, for example, by replacing the coding sequence with human heavy and light chain constant domains in place of the homologous murine sequences (US Patent 4,816,567; Morrison, et al., Proc. Natl Acad . Sci. USA, 81:6851 (1984)), or by covalently attaching to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically, such non-immunoglobulin polypeptides are replaced by the constant domains of an antibody or are replaced by the variable domains of an antigen-combining site of an antibody to create a chimeric bivalent antibody comprising an antigen-combining site with specificity for an antigen and another antigen combining site with specificity for a different antigen.

[0205] The monoclonal antibodies described here may be monovalent, the preparation of which is well known in the art. For example, one method involves recombinant expression of the immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues may be replaced by another amino acid residue or are deleted so as to avoid cross-linking. In vitro methods are also suitable for the preparation of monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be carried out using routine techniques known in the art.

[0206] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Iminothiolate and methyl-4-mercaptobutyrimidate are examples of suitable reagents for this purpose. 3. Recombinant production in prokaryotic cells

a) Vector Construction

[0207] The polynucleotide sequences encoding the antibodies of the present application can be obtained using standard recombinant techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using PCR techniques or a nucleotide synthesizer. Once obtained, the sequences that encode the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. The selection of an appropriate vector will depend primarily on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains several components, depending on its function (heterologous polynucleotide amplification or expression, or both) and its compatibility with the particular host cell in which it resides. Vector components generally include, but are not limited to: an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0208] In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector typically carries a replication site as well as marker sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet) and therefore provides an easy means to identify transformed cells. pBR322, its derivatives or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microbial organism for the expression of endogenous proteins. Examples of pBR322 derivatives used for the expression of particular antibodies are described in detail in Carter et al., US Patent 5,648,237.

[0209] Furthermore, phage vectors that contain replicons and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as GEM™ -11 can be used in preparing a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392.

[0210] The expression vector of the present application may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters generally fall into two classes, inducible and constitutive. The inducible promoter is a promoter that initiates increased levels of cistron transcription under its control in response to changes in culture condition, for example, presence or absence of a nutrient or change in temperature.

[0211] A large number of promoters, recognized by a variety of potential host cells, are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA through restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Both the native promoter sequence and many heterologous promoters can be used to direct amplification and/or expression of target genes. In some embodiments, heterologous promoters are used, as they generally allow for greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0212] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the galactamase and lactose promoter systems, a tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that are functional in bacteria (such as other promoters known from bacteria or phages) are also suitable. Their nucleic acid sequences have been published, thereby allowing a skilled worker to operably link them to cistrons encoding target light and heavy chains (Siebenlist et al. (1980) Cell 20:269) using linkers or adapters to provide all necessary restriction sites.

[0213] In one aspect, each cistron within the recombinant vector comprises a secretion signal sequence component that directs the translocation of expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention must be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequences of heterologous polypeptides, the signal sequence is replaced with a prokaryotic signal sequence selected, for example, from the group consisting of alkaline phosphatase, penicillinase, Ipp, stable leaders of enterotoxin II (STII), LamB, PhoE, PelB, OmpA and MBP. In some embodiments of the present application, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0214] In some embodiments, production of antibodies according to the present application can occur in the cytoplasm of the host cell and therefore does not require the presence of secretion signal sequences within each cistron. In some embodiments, the polypeptide components, such as the polypeptide encoding the VH domain of the first antigen-binding portion optionally fused to the second antigen-binding portion, and the polypeptide encoding the VL domain of the first antigen-binding portion optionally fused to the second antigen-binding portion, and the polypeptide encoding the VL domain of the first antigen-binding portion optionally fused to the second antigen-binding portion, fused to the second antigen-binding moiety, are expressed, folded, and assembled to form functional antibodies within the cytoplasm. Certain host strains (e.g., E. coli trxB- strains) provide cytoplasmic conditions that are favorable for the formation of disulfide bonds, thereby allowing proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0215] The present invention provides an expression system in which the quantitative proportion of expressed polypeptide components can be modulated so as to maximize the yield of secreted and suitably assembled antibodies of the present application. This modulation is accomplished, at least in part, by simultaneous modulation of translational forces for the polypeptide components. A technique for modulating translation strength is disclosed in Simmons et al., US Patent 5,840,523. It uses variants of the translation initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thus providing a convenient means of tuning this factor to the desired expression level of the specific chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes that can alter the amino acid sequence, although silent changes in the nucleic acid sequence are preferred. Changes in TIR may include, for example, changes in the number or spacing of Shine-Dalgarno sequences, along with changes in the signal sequence. One method for generating mutant signal sequences is to generate a "codon bank" at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be achieved by changing the third nucleotide position of each codon; Additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity when making the bank. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS:A Companion to Methods in Enzymol. 4:151-158.

[0216] Preferably, a set of vectors is generated with a range of TIR forces for each cistron therein. This limited set provides a comparison of the expression levels of each chain as well as the yield of desired protein products under various TIR strength combinations. TIR strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. US Patent 5,840,523. Based on the translation strength comparison, the desired individual TIRs are selected to be combined into the expression vector constructs of the present application.

b) Prokaryotic Host Cells.

[0217] Prokaryotic host cells suitable for expressing the antibodies of the present application include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella , Rhizobia, Vitreoscilla, or Paracoccus. In some embodiments, gram-negative cells are used. In some embodiments, E. coli cells are used as hosts for the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D. C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A(nmpc-fepE) degP41 kanR (US Patent 5,639,635). Other strains and derivatives thereof, 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 suitable. These examples are illustrative, not limiting. Methods for constructing derivatives of any of the above-mentioned bacteria, having defined genotypes, are known in the art and are described, for example, in, Bass et al., Proteins, 8:309314 (1990). In general, it is necessary to select appropriate bacteria taking into account the replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as hosts when well-known plasmids, such as pBR322, pBR325, pACYC177, or pKN410, are used to provide the replicon.

[0218] Typically, the host cell must secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may be desirable incorporated into the cell culture.

c) Protein production

[0219] Host cells are transformed with the expression vectors described above and cultivated in conventional nutrient media modified as appropriate to induce promoters, select transformants or amplify genes encoding the desired sequences. Transformation means the introduction of DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or as a chromosomal member. Depending on the host cell used, transformation is done using standard techniques appropriate for those cells. Calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell wall barriers. Another transformation method employs polyethylene glycol/DMSO. Another technique used is electroporation.

[0220] The prokaryotic cells used to produce the antibodies of the present application are cultivated in media known in the art and suitable for culturing the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutritional supplements. In some embodiments, the medium also contains a selection agent, chosen based on the construction of the expression vector, to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growing cells expressing the ampicillin-resistant gene.

[0221] All necessary supplements in addition to carbon, nitrogen and inorganic phosphate sources can also be included in appropriate concentrations, alone or in mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol.

[0222] Prokaryotic host cells are grown at suitable temperatures. For the growth of E. coli, for example, the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably about 30°C. °C. The pH of the medium can be any pH between about 5 and about 9, depending primarily on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, and more preferably about 7.0.

[0223] If an inducible promoter is used in the expression vector of the present application, expression of the protein is induced under conditions suitable for activation of the promoter. In one aspect of the present application, PhoA promoters are used to control transcription of the polypeptides. Accordingly, the transformed host cells are grown in a phosphate-limiting medium for induction. Preferably, the phosphate limiting medium is C. R. A. P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety of other inductors may be used, depending on the vector construct used, as is known in the art.

[0224] The expressed antibodies of the present application are secreted and recovered from the periplasm of the host cells. Protein recovery generally involves disruption of the microorganism, usually by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cellular debris or whole cells can be removed by centrifugation or filtration. Proteins can be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported to the culture medium and isolated therein. The cells can be removed from the culture and the culture supernatant filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods, such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0225] Alternatively, protein production is driven in large quantities by a fermentation process. Several large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations are at least 1000 liters in capacity, preferably about 1000 to 100,000 liters in capacity. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source). Small-scale fermentation generally refers to fermentation in a fermenter that is no more than approximately 100 liters in volumetric capacity and can range from about 1 liter to about 100 liters.

[0226] During the fermentation process, induction of protein expression is typically initiated after cells have been grown under suitable conditions to a desired density, for example, an OD550 of about 180-220, at which stage the cells are in the initial stationary phase. A variety of inductors can be used, depending on the vector construct used, as is known in the art and described above. Cells can be cultured for shorter periods before induction. Cells are generally induced for about 12 to 50 hours, although a longer or shorter induction time may be used.

[0227] To improve the production yield and quality of the antibodies of the present application, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of secreted polypeptides, additional vectors that overexpress chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a cis, trans-peptidylprolyl isomerase with chaperone activity) can be used to cotransform host prokaryotic cells. Chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et al., US Patent 6,083,715; Georgiou et al., US Patent 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.

[0228] To minimize proteolysis of heterologous expressed proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present invention. For example, host cell strains can be modified to make genetic mutations in genes encoding known bacterial proteases, such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some protease-deficient E. coli strains are available and described, for example, in, Joly et al. (1998), supra; Georgiou et al., US Patent 5,264,365; Georgiou et al., US Patent 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0229] E. coli strains deficient for proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can be used as host cells in the expression system encoding the antibodies of the present application.

d) Protein purification

[0230] The antibodies produced here are further purified to obtain preparations that are substantially homogeneous for other assays and uses. Standard protein purification methods known in the art may be used. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE , ammonium sulfate precipitation and gel filtration using, for example, Sephadex G -75.

[0231] In one aspect, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies comprising an Fc region of the present application. Protein A is a 41kD cell wall protein from Staphylococcus aureas that binds with high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth62:1-13. The solid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants not specifically bound to the solid phase. Finally, the antibodies of interest are recovered from the solid phase by elution.

4. Recombinant production in prokaryotic cells

[0232] For eukaryotic expression, the components of the vector generally include, among others, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a termination sequence. transcription.

a) Signal sequence component

[0233] A vector for use in a eukaryotic host can also be an insert that encodes a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The selected heterologous signal sequence is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example the herpes simplex gD signal, are available.

[0234] The DNA for such a precursor region is linked in the reading frame to the DNA encoding the antibodies of the present application.

b) Origin of replication

[0235] Generally, the origin of replication component is not required for mammalian expression vectors (the SV40 origin can normally be used only because it contains the initial promoter).

c) Selection Gene Component

[0236] Expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) provide critical nutrients not available from complex means, for example, the gene encoding D-alanine racemase for Bacilli.

[0237] An example of a selection scheme uses a drug to arrest the growth of a host cell. Cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regime. Examples of such dominant selection use drugs neomycin, mycophenolic acid and hygromycin.

[0238] Another example of selectable markers suitable for mammalian cells are those that allow the identification of cells competent to absorb the nucleic acid encoding the antibodies of the present application, such as DHFR, thymidine kinase, metallothionein-I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0239] For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0240] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with the polypeptide DNA coding sequences, wild-type DHFR protein and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell culture in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, for example, kanamycin, neomycin or G418. See US Patent 4,965,199.

d) Promoter Component

[0241] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the desired polypeptide sequences. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is a CNCAAT region where N can be any nucleotide. The 3' end of most eukaryotes is an AATAAA sequence which may be the signal for the addition of the poly A tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0242] Other promoters suitable for use with prokaryotic hosts include the phoA promoter, the lactation and lactose promoter systems, the alkaline phosphatase promoter, a tryptophan (trp) promoter system and hybrid promoters, such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibodies.

[0243] Polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses, such as polyoma virus, floodplain virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and most preferably simian virus 40 (SV40), from heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, of heat shock promoters, provided that these promoters are compatible with host cell systems.

[0244] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The human cytomegalovirus immediate early promoter is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is disclosed in US Patent 4,419,446. A modification of this system is described in US Patent 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human interferon cDNA in mouse cells under the control of a promoter of herpes simplex virus thymidine kinase. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

e) Enhancer Element Component

[0245] Transcription of a DNA encoding the antibodies of the present application by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, an enhancer from a eukaryotic cellular virus will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancer elements for the activation of eukaryotic promoters. The enhancer can be attached to the vector at a position 5' or 3' to the polypeptide coding sequence, but is preferably located at a 5' site of the promoter.

f) Transcription termination component

[0246] Expression vectors used in eukaryotic host cells (yeast, fungal, insect, plant, animal, human or nucleated cells from other multicellular organisms) also contain sequences necessary for transcription termination and to stabilize the mRNA. Such sequences are commonly available from the 5' and occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain segments of nucleotides transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. A useful transcription termination component is the polyadenylation region of bovine growth hormone. See WO94/11026 and the expression vector disclosed herein.

g) Selection and transformation of host cells

[0247] Host cells suitable for cloning or expressing DNA in the vectors included herein higher eukaryotic cells described herein, including vertebrate host cells. The propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are SV40-transformed monkey kidney line CV1 (COS-7, ATCC CRL 1651); human embryogenic kidney line (293 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)); rat sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980). monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); , ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); 1982)); MRC 5 cells; FS4 cells;

[0248] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants or amplify genes encoding the desired sequences.

h) Cultivation of host cells

[0249] The host cells used to produce the antibodies of the present application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for the cultivation of host cells. Furthermore, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Patent 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or US Patent Re. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (defined as inorganic compounds generally present in final concentrations in the micromolar range), and glucose or an equivalent energy source. 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, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to one skilled in the art.

i) Protein purification

[0250] Using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris, or host cell or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted into the E. coli periplasmic space. In brief, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over the course of approximately 30 min. Cellular debris can be removed by centrifugation. When the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the previous steps to inhibit proteolysis and antibiotics can be included to prevent the growth of accidental contaminants.

[0251] The protein composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies based on human immunoglobulins containing 1, 2 or 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). G protein is recommended for all isotypes of mice and for humans (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is bound is most often agarose, but other matrices are available. Mechanically stable matrices, such as controlled-pore glass or poly(styrene-divinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, Bakerbond ABXTMresin (J. Baker, Phillipsburg, N.J) is useful for purification. Other techniques for protein purification such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, SEPHAROSE™ heparin chromatography, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), Chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0252] After any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4 .5, preferably carried out with low salt concentrations (for example, from about 0-0.25M salt). Immunoconjugates

[0253] In some embodiments, the present application also provides immunoconjugates comprising any of the antibodies (such as sdAbs) described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., toxins proteins, enzymatically active toxins of bacterial, fungal, vegetable or animal origin, or fragments thereof) or radioactive isotopes.

[0254] In some embodiments an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see US Patent 5,208,020, 5,416. 064 and European Patent EP 0 425 235 BI); an auristatin, as monomethylauristatin drug fractions DE and DF (MMAE and MMAF) (see US Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see US 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); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. 16:717-721 (2005); Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); methotrexate; vindesina; a taxane, such as docetaxel, paclitaxel, larotaxel,thesistaxel, and ortataxel; a trichothecene; and CC1065.

[0255] In some embodiments, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, diphtheria A chain. exotoxin (from Pseudomonas aeruginosa), ricin A chain, abren A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gerarnin, mitogillin, restrictocin, fenomycin, enomycin and trichothecenes.

[0256] In some embodiments, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. 211 131 125 90 186 188 153 212 32 212 When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphy studies, for example tc99m or 1123, or a spin tracer for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri ), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0257] Conjugates of an antibody and cytotoxic agent can be produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N- maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniobenzoyl)-ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and bis-active compounds of fluorine (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclotides to antibody. See WO94/11026. The linker may be a “cleavable linker” that facilitates the release of a cytotoxic drug into the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); US Patent 5,208,020) can be used.

[0258] Immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linking 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, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A). Methods and Compositions for Diagnostics and Detection

[0259] 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 "detect" as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample is blood, serum, or other liquid samples of biological origin. In some embodiments, a biological sample comprises a cell or tissue.

[0260] In some embodiments, an anti-BCMA antibody (such as any of the anti-BCMA sdAbs described herein) is provided for use in a diagnostic or detection method. In a further aspect, a method of detecting the presence of BCMA in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of BCMA protein in a biological sample. In certain embodiments, the BCMA is human BCMA. In certain embodiments, the method comprises contacting the biological sample with an anti-BCMA antibody as described herein under conditions permissive for binding of the anti-BCMA antibody to BCMA, and detecting whether a complex is formed between the anti-BCMA antibody. and BCMA. This method can be an in vitro or in vivo method. In some embodiments, an anti-BCMA antibody is used to select individuals eligible for therapy with an anti-BCMA antibody, for example where BCMA is a biomarker for patient selection.

[0261] In certain embodiments, labeled anti-BCMA sdAbs are provided. Tags include, but are not limited to, tags or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive tags), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g. , through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g. example, firefly luciferase and bacterial luciferase (US Patent 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g. example, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, together with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin tags, bacteriophage tags, stable free radicals and the like. III. Chimeric antigen receptors

[0262] One aspect of the present application provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising one or more single-domain antibodies (such as VHHs). Any of the anti-BCMA sdAbs described in Section II can be used in the CARs described here. Exemplary structures of CARs are shown in FIGs. 15A-15D.

[0263] In some embodiments, there is provided a CAR that targets BCMA (also referred to herein as "BCMA CAR") comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human or humanized. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3r. In some embodiments, the primary intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 -H3, CD83 Ligands and combinations thereof. In some embodiments, the BCMA CAR also comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first co-stimulatory signaling domain derived from CD28 , a second costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3r. In some embodiments, the BCMA CAR is monospecific. In some embodiments, the CAR BCMA is monovalent.

[0264] In some embodiments, there is provided a BCMA CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52; and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human or humanized. In some embodiments, the anti-BCMA sdAb comprises a VHH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 115-152. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of a immune effector cell (such as T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3α. In some embodiments, the primary intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 -H3, CD83 Ligands and combinations thereof. In some embodiments, the BCMA CAR also comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first co-stimulatory signaling domain derived from CD28 , a second costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3r. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137-derived costimulatory signaling domain, and a primary intracellular signaling domain derived from CD3. In some embodiments, the BCMA CAR is monospecific. In some embodiments, the CAR BCMA is monovalent.

[0265] In some embodiments, there is provided a BCMA CAR comprising a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity for the amino acid sequence selected from the group consisting of SEQ ID NO:216-256 and 298-335. In some embodiments, a BCMA CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:216-256 and 298-335 is provided. Also provided is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:216-256 and 298-335.

[0266] In some embodiments, an isolated nucleic acid encoding any of the BCMA CARs provided herein is provided. In some embodiments, there is provided an isolated nucleic acid having at least approximately any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99% or 100% sequence identity for a nucleic acid sequence selected from the group consisting of SEQ ID NOs:257-297 and 336-373. In some embodiments, an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 257-297 and 336-373 is provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is an RNA. In some embodiments, a vector comprising any 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 non-viral vector. Exemplary monovalent BCMA CARs are shown in Table 4 below. Table 4. Exemplary monovalent BCMA CARs.

Multivalent chimeric antigen receptors

[0267] The present application also provides multivalent CARs that have two or more (such as about any one of 2, 3, 4, 5, 6 or more) binding moieties that specifically bind to an antigen, such as BCMA. In some embodiments, one or more of the binding moieties are antigen-binding fragments. In some embodiments, one or more of the binding moieties comprises single-domain antibodies. In some embodiments, one or more of the binding moieties are derived from camelid antibodies. In some embodiments, one or more of the binding moieties are derived from a four-chain antibody. In some embodiments, one or more of the binding moieties are scFvs. In some embodiments, one or more of the binding moieties are derived from human antibodies. In some embodiments, one or more of the binding moieties are polypeptide ligands or other non-antibody polypeptides that specifically bind to the antigen. In some embodiments, the multivalent CAR is monospecific, that is, the multivalent CAR targets a single antigen and comprises two or more binding sites for the single antigen. In some embodiments, the multivalent CAR is monospecific, that is, the multivalent CAR targets more than one antigen and the multivalent CAR comprises two or more binding sites for at least one antigen. Binding moieties specific to the same antigen can bind to the same epitope of the antigen (i.e., “monoepitope CAR”) or bind to different epitopes (i.e., “multiepitope CAR” such as biepitope CAR or triepitope CAR) of the antigen. Specific binding sites for the same antigen may comprise the same or different sdAbs.

[0268] In some embodiments, the present application provides a multivalent (such as bivalent, trivalent, or higher number of valences) chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a plurality ( such as at least about any one of 2, 3, 4, 5, 6 or more) of binding moieties that specifically bind to an antigen (such as a tumor antigen); (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY -ESO-1 MAGE A3 and glycolipid F77.

[0269] In some embodiments, the present application provides a multivalent (such as bivalent, trivalent, or higher number of valences) chimeric antigen receptor comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a plurality ( such as at least about any one of 2, 3, 4, 5, 6 or more) single domain antibodies (sdAb) 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.

[0270] In some embodiments, the present application provides a multivalent chimeric antigen receptor (such as bivalent, trivalent or higher number of valences) comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising a first moiety binding moiety that specifically binds to a first epitope of an antigen (such as a tumor antigen), and a second binding moiety that specifically binds to a second epitope of the antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY -ESO-1 MAGE A3 and glycolipid F77. In some embodiments, the first binding moiety is an sdAb and the second binding moiety is derived from a human antibody (e.g., a scFv). In some embodiments, the first binding moiety is an sdAb and the second binding moiety is a polypeptide ligand. In some embodiments, the first epitope is the same as the second epitope. In some embodiments, the first epitope is different from the second epitope. In some embodiments, the multivalent CAR specifically binds to two different epitopes on an antigen. In some embodiments, the multivalent CAR specifically binds to three or more different epitopes on an antigen.

[0271] In some embodiments, the present application provides a multivalent chimeric antigen receptor (such as bivalent, trivalent or higher number of valences) comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising a first sdAb that specifically binds to a first epitope of an antigen (such as a tumor antigen), and a second sdAb that specifically binds to a second epitope of the antigen; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different. In some embodiments, the antigen is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY -ESO-1 MAGE A3 and glycolipid F77.

[0272] In some embodiments, the binding moieties such as sdAbs (including the plurality of sdAbs, or the first sdAb and/or the second sdAb) are camelid, chimeric, human or humanized. In some embodiments, the binding moieties or sdAbs are fused together through peptide bonds or peptide linkers. In some embodiments, each peptide linker is no more than about 50 (such as no more than any of 35, 25, 20, 15, 10, or 5) amino acids in length. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3r. In some embodiments, the primary intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 -H3, CD83 Ligands and combinations thereof. In some embodiments, the multivalent CAR also comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multivalent CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3r. In some embodiments, the multivalent CAR is monospecific. In some embodiments, the multivalent CAR is multispecific, such as bispecific.

[0273] The multivalent CARs described here may be especially suitable for targeting multimeric antigens through synergistic binding by different antigen binding sites, or for increasing binding affinity or avidity to the antigen. Any of the anti-BCMA sdAbs described herein can be used in the extracellular antigen-binding domain of the multivalent CARs described herein. A list of exemplary multivalent BCMA CARs, exemplary sequences, constructs and vectors thereof are shown in Table 5.

[0274] In some embodiments, there is provided a multivalent CAR that targets BCMA comprising: (a) an extracellular antigen-binding domain comprising a plurality (such as at least about any one of 2, 3, 4 or more) of a BCMA-binding moiety (e.g., an anti-BCMA sdAb); (b) a transmembrane domain; and (c) an intracellular signaling domain. Any of the anti-BCMA sdAbs can be used to construct the multivalent BCMA CAR. In some embodiments, the extracellular antigen-binding domain specifically binds to a single BCMA epitope, and these CARs are referred to herein as monoepitope multivalent BCMA CARs.

[0275] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a plurality (such as at least about any one of 2, 3, 4 or more) of an sdAb anti-BCMA; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:39, and a CDR3 comprising the amino acid sequence of SEQ ID NO:77.

[0276] In some embodiments, there is provided a multivalent BCMA CAR (also referred to herein as "multivalent multiepitope CAR") comprising: (a) an extracellular antigen-binding domain comprising at least two (such as any of 2, 3, 4 or more) BCMA-binding moieties; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the at least two BCMA-binding moieties specifically bind to at least two different epitopes on BCMA. In some embodiments, the extracellular antigen-binding domain comprises a first BCMA-binding moiety and a second BCMA-binding moiety. In some embodiments, the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is derived from a human antibody (e.g., a scFv). In some embodiments, the first BCMA-binding moiety is an sdAb and the second BCMA-binding moiety is a BCMA polypeptide ligand. In some embodiments, the first anti-BCMA binding moiety and/or the second BCMA binding moiety specifically binds to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs:388-394. In some embodiments, the first BCMA-binding moiety specifically binds to an epitope derived from SEQ ID NO:389 and/or 390. In some embodiments, the second BCMA-binding moiety specifically binds to an epitope derived from SEQ ID NO:391 and/or 392.

[0277] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first anti-BCMA sdAb and the second anti-BCMA sdAb specifically bind to different epitopes on BCMA. Any of the anti-BCMA sdAbs can be used to construct the multivalent BCMA CAR. In some embodiments, the first anti-BCMA sdAb and/or the second anti-BCMA sdAb specifically binds to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs:388-394. In some embodiments, the first anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NO: 389 and/or 390. In some embodiments, the second anti-BCMA sdAb specifically binds to an epitope derived from SEQ ID NO: 391 and/or 392.

[0278] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:3, a CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; and wherein the second anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:10, a CDR2 comprising the amino acid sequence of SEQ ID NO:48, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the first anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:117. In some embodiments, the second anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:124. In some embodiments, the first anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:124. In some embodiments, the second anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:117.

[0279] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:10, a CDR2 comprising the amino acid sequence of SEQ ID NO:48, and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; and wherein the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:49 and a CDR3 comprising the amino acid sequence of SEQ ID NO:87 . In some embodiments, the first anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:124. In some embodiments, the second anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:125.

[0280] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:45, and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; and wherein the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:49 and a CDR3 comprising the amino acid sequence of SEQ ID NO:87 . In some embodiments, the first anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:121. In some embodiments, the second anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:125.

[0281] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:15, a CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; and wherein the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:56 and a CDR3 comprising the amino acid sequence of SEQ ID NO:94 . In some embodiments, the first anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:129. In some embodiments, the second anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:132.

[0282] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:18, a CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; and wherein the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:20, a CDR2 comprising the amino acid sequence of SEQ ID NO:58, and a CDR3 comprising the amino acid sequence of SEQ ID NO:96 . In some embodiments, the first anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:132. In some embodiments, the second anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:134.

[0283] In some embodiments, a multivalent BCMA CAR is provided, comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb and a second anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:20, a CDR2 comprising the amino acid sequence of SEQ ID NO:58, and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; and wherein the anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:28, a CDR2 comprising the amino acid sequence of SEQ ID NO:66, and a CDR3 comprising the amino acid sequence of SEQ ID NO:104 . In some embodiments, the first anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:134. In some embodiments, the second anti-BCMA sdAb comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:142.

[0284] 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 ligand. In some embodiments, the peptide linker is no more than about 50 (such as no more than any of 35, 25, 20, 15, 10, or 5) amino acids in length. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3α. In some embodiments, the primary intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 -H3, CD83 Ligands and combinations thereof. In some embodiments, the multivalent BCMA CAR also comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multivalent BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3. In some embodiments, the multivalent CAR BCMA is bivalent. In some embodiments, the multivalent CAR BCMA is trivalent. In some embodiments, the multivalent BCMA CAR specifically binds to two different epitopes on a BCMA. In some embodiments, the multivalent CAR BCMA specifically binds to three or more different epitopes on a BCMA.

[0285] In some embodiments, there is provided a multivalent BCMA CAR comprising a polypeptide having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% sequence identity for the amino acid sequence selected from the group consisting of SEQ ID NO:298-335. In some embodiments, a multivalent BCMA CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:298-335 is provided. Also provided is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:298-335.

[0286] In some embodiments, an isolated nucleic acid encoding any of the multivalent BCMA CARs provided herein is provided. In some embodiments, there is provided an isolated nucleic acid having at least approximately any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99% or 100% sequence identity for a nucleic acid sequence selected from the group consisting of SEQ ID NOs:336-373. In some embodiments, an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:336-373 is provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is an RNA. In some embodiments, a vector comprising any of the nucleic acids encoding the multivalent BCMA CARs described above is provided. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector. Exemplary multivalent BCMA CARs are shown in Table 5 below. Multispecific chimeric antigen receptors

[0287] The present application further provides multispecific chimeric antigen receptors that target two or more (e.g., about any one of 2, 3, 4, 5, 6 or more) different antigens. In some embodiments, the multispecific CAR has a single antigen-binding site for each antigen. In some embodiments, the multispecific CARs have more than two binding sites for at least one antigen. Each antigen binding site can comprise an sdAb. For example, in some embodiments, the multispecific CAR is a bispecific CAR comprising an extracellular antigen-binding domain comprising two different sdAbs each specifically binding to an antigen. In some embodiments, the multispecific CAR is a trispecific CAR comprising an extracellular antigen binding domain comprising three different SdAbs each specifically binding to an antigen.

[0288] In some embodiments, there is provided a multispecific (such as bispecific) chimeric antigen receptor (CAR) comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first single-domain antibody (sdAb) that is binds specifically 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, in which 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 is camelid, chimeric, human or humanized. In some embodiments, the first sdAb and the second sdAb are fused to each other through a peptide bond or a peptide linker. In some embodiments, the peptide linker is no more than about 50 (such as no more than any of 35, 25, 20, 15, 10, or 5) amino acids in length. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3. In some embodiments, the primary intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 -H3, CD83 Ligands and combinations thereof. In some embodiments, the multispecific CAR also comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multispecific CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3r. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a CD28-derived costimulatory signaling domain, and a primary intracellular signaling domain derived from CD3. Extracellular antigen-binding domain

[0289] The extracellular antigen-binding domain of the CARs described herein comprises one or more single-domain antibodies (such as any of 1, 2, 3, 4, 5, 6 or more) binding moieties, such as sdAbs. In some embodiments, the one or more binding moieties are antibodies or antigen-binding fragments thereof. In some embodiments, the one or more binding moieties are derived from four-chain antibodies. In some embodiments, the one or more binding moieties are derived from camelid antibodies. In some embodiments, the one or more binding moieties are derived from human antibodies. In some embodiments, the one or more binding moieties are non-antibody binding proteins, such as polypeptide ligands or engineered proteins that bind to an antigen. The linker moieties can be fused to each other directly through peptide bonds, or through peptide linkers.

1. Single-domain antibodies

[0290] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising one or more sdAbs. sdAbs can be of the same type of different origins and of the same or different sizes. Exemplary sdAbs include, but are not limited to, heavy chain variable domains from heavy chain only antibodies (e.g., VHH or VNAR), binding molecules naturally devoid of light chains, single domains (such as VH or VL) derived from antibodies conventional 4-chain antibodies, humanized heavy chain antibodies, human sdAbs produced by transgenic mice or rats expressing human heavy chain segments and engineered domains, and single-domain scaffolds other than those derived from antibodies. Any sdAbs known in the art or developed by the inventors, including the sdAbs described in Section II of the present application, can be used to construct the CARs described herein. sdAbs can be derived from any species, including but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cattle. The single domain antibodies contemplated herein also include naturally occurring sdAb molecules from species other than Camelidae and sharks.

[0291] In some embodiments, the sdAb is derived from a naturally occurring single-domain antigen-binding molecule known as a heavy chain antibody devoid of light chains (also referred to herein as "heavy chain only antibodies"). Such single-domain molecules are disclosed in WO 94/04678 and Hamers-Caserman, C. et al. (1993) Nature 363:446-448, for example. For reasons of clarity, the variable domain derived from a heavy chain molecule naturally lacking a light chain is known herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in species of Camelidae, for example, camel, llama, vicuna, dromedary, alpaca and guanaco. Species other than Camelidae can produce heavy chain molecules, naturally lacking a light chain, and such VHHs are within the scope of the present application.

[0292] Camelid VHH molecules are about 10 times smaller than IgG molecules. They are unique polypeptides and can be very stable, resisting extreme pH and temperature conditions. Furthermore, they may be resistant to the action of proteases, which is not the case with conventional 4-chain antibodies. Furthermore, in vitro expression of VHHs produces high-yield and appropriately folded functional VHHs. Furthermore, antibodies generated in camelids can recognize epitopes other than those recognized by antibodies generated in vitro through the use of antibody libraries or through immunization of mammals other than camelids (see, for example, WO9749805). As such, multispecific or multivalent CARs comprising one or more VHH domains can interact more efficiently with targets than multispecific or multivalent CARs comprising antigen-binding fragments derived from conventional 4-chain antibodies. Since VHHs are known to bind to "unusual" epitopes such as pits or grooves, the affinity of CARs comprising such VHHs may be more suitable for therapeutic treatment than conventional multispecific polypeptides.

[0293] In some embodiments, the sdAb is derived from a variable region of the immunoglobulin found in cartilaginous fish. For example, the sdAb may be derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in shark serum. Methods of producing single-domain molecules derived from a NAR variable region ("IgNARs") are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0294] In some embodiments, the sdAb is recombinant, CDR-grafted, humanized, camelized, deimmunized, and/or generated in vitro (e.g., selected by phage display). In some embodiments, the amino acid sequence of the framework regions can be altered by "camelizing" specific amino acid residues in the framework regions. Camelization refers to the replacement or exchange of one or more amino acid residues in the amino acid sequence of a (naturally occurring) VH domain of a conventional 4-chain antibody with one or more of the amino acid residues occurring in the corresponding position(s) in a VHH domain of a heavy chain antibody. This may be carried out in a manner known per se, which will be clear to the skilled artisan, for example based on the further description described herein. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and/or are present at the VH-VL interface and/or at so-called Camelidae trademark residues as defined herein (see, for example, WO 94 /04678, Davies and Riechmann FEBS Letters 339:285-290, 1994; Davies and Riechmann Protein Engineering 9 (6):531-537, 1996; Muyldermans J. Immunol. 231:25-38, 1999).

[0295] In some embodiments, the sdAb is a human sdAb produced by transgenic mice or rats that express human heavy chain segments. See, for example, US20090307787A1, US Patent 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794. In some embodiments, the sdAb is affinity matured.

[0296] In some embodiments, naturally occurring VHH domains against a given antigen or target can be obtained from libraries (naïve or immune) of Camelid VHH sequences. Such methods may or may not involve scanning such a library using said antigen or target, or at least a part, fragment, antigenic determinant or epitope thereof using one or more scanning techniques known per se. Such libraries and techniques are, for example, described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from VHH (naïve or immune) libraries can be used, such as VHH libraries obtained from VHH (naïve or immune) libraries by techniques such as random mutagenesis and/or CDR mixing, as by example described in WO 00/43507.

[0297] In some embodiments, sdAbs are generated from conventional four-chain antibodies. See, for example, EP 0 368 684, Ward et al. (Nature 1989 Oct. 12; 341 (6242):544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; WO 06/030220; and WO 06/003388.

2. Antigens

[0298] The antigen(s) targeted by the CARs of the present application are cell surface molecules. Binding moieties (such as sdAbs) can be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen (such as the first antigen and/or the second antigen) is a tumor antigen. In some embodiments, multispecific CARs target two or more tumor antigens. In some embodiments, the tumor antigen is associated with a B-cell malignancy. Tumors express a number of proteins that can serve as a target antigen for an immune response, particularly T-cell-mediated immune responses. be antigens on a single cell or diseased antigens that are expressed on different cells that contribute to the disease. The antigens targeted by CAR may be directly or indirectly involved in diseases.

[0299] Tumor antigens are proteins that are produced by tumor cells that can provoke an immune response, particularly T cell-mediated immune responses. The selection of the target antigen of the invention will depend on the particular type of cancer to be treated. Exemplary tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX , human telomerase reverse transcriptase RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2/neu, survivin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0300] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a series of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the HER2/Neu/ErbB-2 oncogene. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotypic immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B-cell lymphoma.

[0301] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur in other cells in the body. A TAA-associated antigen is not exclusive to a tumor cell and, instead, is also expressed on a normal cell under conditions that fail to induce a state of immunological tolerance to the antigen. Antigen expression in the tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels. high in tumor cells.

[0302] Other non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens, such as MART-1/MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE -1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus EBVA antigens and the human papillomavirus (HPV) E6 and E7 antigens. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17. 1, NuMa, K ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG , BCA225, BTAA, CA 125, CA 15-3\CA 27. 29\BCAA, CA 195, CA 242, CA- 50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA- 50, MG7-Ag, MOV18, NB/70K, NY-CO- 1, RCAS 1, SDCCAG16, TA- 90 binding protein\Mac-2\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0303] In some embodiments, the antigen (such as the first antigen and/or the second antigen) is selected from the group consisting of CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL -13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1 MAGE A3 and glycolipid F77. 3. Peptide ligands

[0304] The various binding moieties (such as sdAbs) in the multispecific or multivalent CARs described herein can be fused together via peptide linkers. In some embodiments, the binding moieties (such as sdAbs) are directly fused together without any peptide linkers. The peptide ligands that bind different binding moieties (such as sdAbs) can be the same or different. Different domains of CARs can also be fused to each other via peptide linkers.

[0305] Each peptide linker in a CAR may have the same or different length and/or sequence depending on the structural and/or functional characteristics of the sdAbs and/or various domains. Each peptide ligand can be selected and optimized independently. The length, degree of flexibility and/or other properties of the peptide linker(s) used in CARs may have some influence on the properties, including, but not limited to affinity, specificity or avidity for a or more particular antigens or epitopes. For example, longer peptide linkers can be selected to ensure that two adjacent domains do not sterically interfere with each other. For example, in a multivalent or multispecific CAR of the present application comprising sdAbs directed against a multimeric antigen, the length and flexibility of the peptide linkers are preferably such that they allow each sdAb in the multivalent CAR to bind to the antigenic determinant on each of the subunits. of the multimer. In some embodiments, a short peptide linker may be disposed between the transmembrane domain and the intracellular signaling domain of a CAR. In some embodiments, a peptide linker comprises flexible residues (such as glycine and serine) so that adjacent domains are free to move relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker.

[0306] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 25 30, 35, 40, 50, 75, 100 or more amino acids in length. In some embodiments, the peptide linker is no more than any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acids in length. In some embodiments, the length of the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids. , about 10 amino acids to about 25 amino acids long, about 5 amino acids to about 30 amino acids long, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids long, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0307] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain antibodies can only be used as the linker. See, for example, WO1996/34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is a number whole of at least one), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence GGGGS (SEQ ID NO:208), (GGGGS)2 (SEQ ID NO:209), (GGGS)4 (SEQ ID NO:210), GGGGSGGGGSGGGGGGSGSGGGGS (SEQ ID NO: :211), GGGGSGGGGSGGGGGGSGSGGGGSGGGGSGGGGS (SEQ ID NO:212), (GGGGS)3 (SEQ ID NO:213), (GGGGS)4 (SEQ ID NO:214), or (GGGGS)3 (SEQ ID NO:215).

Transmembrane domain

[0308] The CARs of the present application comprise a transmembrane domain that can be directly or indirectly fused with the extracellular antigen binding domain. The transmembrane domain can be derived from a natural source or a synthetic source. As used herein, a "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Compatible transmembrane domains for use in the CARs described here can be obtained from a natural protein. Alternatively, it may be a synthetic, non-naturally occurring protein segment, for example, a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0309] Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, transmembrane domains can form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Furthermore, transmembrane domains can also be classified according to the topology of the transmembrane domain, including the number of passes the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins pass through the cell membrane once, and multiple-pass membrane proteins pass through the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 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 segment(s) relative to the interior and exterior of the cell. Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the cell's lipid bilayer and the C-terminus of the protein is present on the cytoplasmic side. , Type II membrane proteins also have a single membrane-spanning region, but are oriented so that the C-terminus of the protein is present on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple membrane-spanning segments and can be subsequently subclassified based on the number of transmembrane segments and the location of the N- and C-termini.

[0310] In some embodiments, the transmembrane domain of the CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains of multiple-pass membrane proteins may also be compatible for use in the CARs described herein . Multipass membrane proteins may comprise a complex of (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure. Preferably, the N-terminus and C-terminus of a multipass membrane protein are present on opposite sides of the lipid bilayer, for example, the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the terminus of the protein is present on the extracellular side.

[0311] In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a 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 (CDl la, 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 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 CD8a, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0312] In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:194. In some embodiments, the transmembrane domain of CD28 is encoded by the nucleic acid sequence of SEQ ID NO:203.

[0313] In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is a CD8α transmembrane domain comprising the amino acid sequence of SEQ ID NO:193. In some embodiments, the transmembrane domain of CD8α is encoded by the nucleic acid sequence of SEQ ID NO:202.

[0314] The transmembrane domains for use in the CARs described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in US Patent 7,052,906 B1 and PCT Publication WO 2000/032776 A2, the relevant disclosures of which are incorporated herein by reference.

[0315] The transmembrane domain may comprise a transmembrane region and a cytoplasmic region located on the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may comprise three or more amino acids and in some embodiments helps orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain comprises the amino acids arginine, serine and lysine.

[0316] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In preferred embodiments, the transmembrane domain of the CAR comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises primarily hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a polyleucine-alanine sequence. Hydropathy, or hydrophobic or hydrophilic characteristics of a protein segment or protein, can be evaluated by any method known in the art, for example, Kyte and Doolittle hydropathy analysis.

Intracellular signaling domain

[0317] The CARs of the present application comprise an intracellular signaling domain. The intracellular signaling domain is responsible for the activation of at least one of the normal ephemeral functions of the immune effector cell expressing CARs. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell, for example, may be cytolytic activity or auxiliary activity including the secretion of cytokines. Thus, the term "cytoplasmic signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. Although the entire cytoplasmic signaling domain can generally be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the cytoplasmic signaling domain is used, such a truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. Therefore, the term cytoplasmic signaling domain means that it includes any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.

[0318] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. "Primary intracellular signaling domain" refers to the cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. An "ITAM", as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif may comprise two repeats of the YxxL/I amino acid sequence separated by 6-8 amino acids, where each x is independently any amino acid, producing the conserved YxxL/Ix (6-8) YxxL/I motif. ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM upon activation of the signaling molecule. ITAMs can also function as docking sites for other proteins involved in signaling pathways. Exemplary primary cytoplasmic signaling sequences containing ITAM include those derived from CD3 r, FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d.

[0319] In some embodiments, the primary intracellular signaling domain is derived from CD3 r. In some embodiments, the intracellular signaling domain consists of the CD3 r cytoplasmic signaling domain. In some embodiments, the primary intracellular signaling domain is a wild-type CD3 cytoplasmic signaling domain. In some embodiments, the primary intracellular signaling domain of wild-type CD3 r comprises the amino acid sequence of SEQ ID NO:197. In some embodiments, the primary intracellular signaling domain is a functional mutant of the CD3 r cytoplasmic signaling domain containing one or more mutations, such as Q65K. In some embodiments, the primary intracellular signaling domain of the mutant CD3 r comprises the amino acid sequence of SEQ ID NO:198. In some embodiments, the primary intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 206 or 207.

Costimulatory signaling domain

[0320] Many immune effector cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, in addition to activating effector functions of the cell. In some embodiments, the CAR comprises at least one costimulatory signaling domain. The term "costimulatory signaling domain" as used herein refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response, such as an effector function. The costimulatory signaling domain of the chimeric receptor described herein may be a cytoplasmic signaling domain from a costimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils or eosinophils. "Costimulatory signaling domain" may be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival.

[0321] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains (such as, for example, any of 2, 3, 4 or more). In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domains, e.g., two copies of the CD28 costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (such as the CD3 r cytoplasmic signaling domain) and one or more co-stimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (such as the CD3 r cytoplasmic signaling domain) are fused together via optional peptide linkers. The primary intracellular signaling domain and one or more co-stimulatory signaling domains may be arranged in any suitable order. In some embodiments, one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as the cytoplasmic signaling domain of CD3). Multiple co-stimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0322] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival and/or cytotoxicity . The costimulatory signaling domain of any costimulatory molecule may be compatible for use in the CARs described herein. The type(s) of costimulatory signaling domain is selected based on factors such as the type of immune effector cells in which the effector molecules would be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g. ADCC effect). Examples of costimulatory signaling domains for use in CARs may be the cytoplasmic signaling domain of costimulatory proteins, including, without limitation, members of the B7/CD28 family (e.g., B7-1/CD80, B7-2/CD86, B7- H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD- 1, PD-L2/B7- DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30 Ligand /TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14, Lymphotoxin-alpha/TNF-beta, OX40/TNFRSF4, OX40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, and TNF RII/TNFRSF1B); members of the SLAM family (e.g., 2B4/CD244/SLAMF4, BLAME/SLAMF8, CD2, CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB- A/SLAMF6, and SLAM/CD150); and any other costimulatory molecules such as CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200, CD300a/LMIR1, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM- 1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, TIM-1/KIM-1/HAVCR, TIM -4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C.

[0323] In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen 1 (LFA-1) , CD2, CD7, LUZ, NKG2C, B7-H3 and ligands that bind especially to CD83.

[0324] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises a CD3 cytoplasmic signaling domain and a CD28 co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:195. In some embodiments, the CD28 costimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO:204.

[0325] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises a CD3 cytoplasmic signaling domain and a CD137 co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a CD137 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:196. In some embodiments, the costimulatory signaling domain of CD137 is encoded by the nucleic acid sequence of SEQ ID NO:205.

[0326] In some embodiments, the intracellular signaling domain in the CAR of the present application comprises a CD28 costimulatory signaling domain and a CD137 costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3 cytoplasmic signaling domain, a CD28 costimulatory signaling domain, and a CD137 costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising from the N-terminus to the C-terminus: a CD28 costimulatory signaling domain, a CD137 costimulatory signaling domain, and a CD3 cytoplasmic signaling domain. In some embodiments, the CD28 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:195. In some embodiments, the CD137 costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:196.

[0327] Also within the scope of the present disclosure are variants of any of the costimulatory signaling domains described herein, such that the costimulatory signaling domain is capable of modulating the immune response of the immune cell. In some embodiments, the costimulatory signaling domains comprise up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to a wild-type counterpart. Such co-stimulatory signaling domains comprising one or more amino acid variations may be referred to as variants. Mutation of amino acid residues of the costimulatory signaling domain can result in increased signaling transduction and enhanced stimulation of immune responses relative to costimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the costimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to costimulatory signaling domains that do not comprise the mutation.

Hinge region

[0328] The CARs of the present application may comprise a hinge domain that is located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is usually found between two domains of a protein and can allow for protein flexibility and movement of one or both domains relative to each other. It is possible to use any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule.

[0329] The hinge domain may contain about 10-100 amino acids, for example, about any one of 15-75 amino acids, 20-50 amino acids or 30-60 amino acids. In some embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 amino acids in length.

[0330] In some embodiments, the hinge domain is a hinge domain of a natural protein. The hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a natural protein and 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 CD8α hinge domain, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the CD8α hinge domain. In some embodiments, the CD8α hinge domain comprises the amino acid sequence of SEQ ID NO:192. In some embodiments, the CD8α hinge domain is encoded by the nucleic acid sequence of SEQ ID NO:201.

[0331] The hinge domains of antibodies, such as IgG, IgA, IgM, IgE or IgD antibodies, are also compatible for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, the hinge domain is the hinge domain that joins the CH1 and CH2 constant domains of an antibody. In some embodiments, the hinge domain is from an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

[0332] Non-natural peptides can also be used as hinge domains for the chimeric receptors described here. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, where x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.

Signal peptide

[0333] The CARs of the present application may comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, signal peptides are peptide sequences that direct a polypeptide to the desired location in a cell. In some embodiments, the signal peptide directs the effector molecule into the secretory pathway of the cell and will allow integration and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including naturally occurring protein signal sequences or non-naturally occurring synthetic signal sequences, that are compatible for use in the CARs described herein will be apparent to one skilled in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSFα receptor, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO:191. In some embodiments, the CD8α signal peptide is encoded by the nucleic acid sequence of SEQ ID NO: 199 or 200.

IV. Engineered immune effector cells

[0334] Furthermore, host cells (such as immune effector cells) comprising any of the CARs described herein are provided in the present application.

[0335] Thus, in some embodiments, there is provided an engineered immune effector cell (such as a T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety that specifically binds to a first BCMA epitope, and a second BMCA-binding moiety that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different.

[0336] In some embodiments, there is provided an engineered immune effector cell (such as a T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb that specifically binds to a first BCMA epitope, and a second anti-BMCA sdAb that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different. In some embodiments, the first anti-BCMA sdAb and/or the second anti-BCMA sdAb is camelid, chimeric, human or humanized. In some embodiments, the first anti-BCMA and the second anti-BCMA are fused to each other via a peptide bond or a peptide linker. In some embodiments, the peptide linker is no more than about 50 (such as no more than any of 35, 25, 20, 15, 10, or 5) amino acids in length. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3r. In some embodiments, the primary intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 -H3, CD83 Ligands and combinations thereof. In some embodiments, the multivalent CAR also comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the multivalent CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137-derived costimulatory signaling domain, and a primary intracellular signaling domain derived from CD3r. In some embodiments, the engineered immune effector cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell. In some embodiments, the engineered immune effector cell is autologous. In some embodiments, the engineered immune effector cell is allogeneic.

[0337] In some embodiments, there is provided an engineered immune effector cell (such as a T cell) comprising a BCMA CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52; and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114. In some embodiments, the extracellular antigen-binding domain comprises at least two anti-BCMA sdAbs. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human or humanized. In some embodiments, the anti-BCMA sdAb comprises a VHH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:115-152. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3. In some embodiments, the primary intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7 -H3, CD83 Ligands and combinations thereof. In some embodiments, the BCMA CAR also comprises a hinge domain (such as a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the BCMA CAR further comprises a signal peptide (such as a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first co-stimulatory signaling domain derived from CD28 , a second costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3. In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus: a CD8α signal peptide, the extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a CD137-derived costimulatory signaling domain, and a primary intracellular signaling domain derived from CD3r. In some embodiments, the BCMA CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:216-256, 298-335. In some embodiments, the engineered immune effector cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell. In some embodiments, the engineered immune effector cell is autologous. In some embodiments, the engineered immune effector cell is allogeneic.

[0338] Engineered immune effector cells comprising (or expressing) two or more different CARs are also provided. Any two or more CARs described herein can be expressed in combination. CARs can target different antigens, thus providing synergistic or additive effects. Because single-domain antibodies in the extracellular antigen-binding domains of CARs have only single-antigen variable chains (such as heavy chains), these CAR-expressing cells do not exhibit variable chain mispairing problems as seen in engineered immune effector cells. coexpressing two or more scFv-based CARs. Exemplary engineered immune effector cells that coexpress two VHH-based CARs are illustrated in FIG. 15E. One skilled in the art would recognize that CARs based on other sdAbs or having other structures, as described herein, can also be coexpressed in engineered immune effector cells. The two or more CARs can be encoded in the same vector or different vectors.

[0339] The engineered immune effector cell may further express one or more therapeutic and/or immunomodulatory proteins, such as immune checkpoint inhibitors. See, for example, International Patent Applications PCT/CN2016/073489 and PCT/CN2016/087855, which are incorporated herein by reference in their entirety.

Vectors

[0340] The present application provides vectors for cloning and expressing any of the CARs described herein. In some embodiments, the vector is suitable for replication and integration into eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vector, retroviral vectors, vaccinia vector, herpes simplex viral vector and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.

[0341] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cell in vitro or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying the immunomodulator coding sequence (such as immune checkpoint inhibitor) and/or self-inactivating lentiviral vectors carrying CARs can be packaged with protocols known in the art. The resulting lentiviral vectors can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art. Retrovirus-derived vectors such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have little immunogenicity and can transduce non-proliferating cells.

[0342] In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a transposon, such as a Sleeping Beauty (SB) transposon system, or a PiggyBac transposon system. In some embodiments, the vector is a polymer-based non-viral vector, including, for example, poly(lactic-coglycolic acid) (PLGA) and polylactic acid (PLA), poly(ethylene imine) (PEI) and dendrimers. In some embodiments, the vector is a non-viral vector based on cationic lipids, such as a cationic liposome, lipid nanoemulsion, and solid lipid nanoparticle (SLN). In some embodiments, the vector is a peptide-based gene non-viral vector, such as poly-L-lysine. Any of the known non-viral vectors suitable for genome editing can be used to introduce CAR-encoding nucleic acids into engineered immune effector cells. See, for example, Yin H. et al. Nature Rev. Genetics (2014) 15:521–555; Aronovich EL et al. “The Sleeping Beauty transposon system: a non-viral vector for gene therapy.” Hum. Mol. Genet. (2011) R1:R14-20; and Zhao S. et al. “PiggyBac transposon vectors: the tools of the human gene editing.” Transl. Lung Cancer Res. (2016) 5(1):120-125, which are incorporated herein by reference. In some embodiments, any one or more of the nucleic acids encoding a CAR are introduced into engineered immune effector cells by a physical method, including, but not limited to, electroporation, sonoporation, photoporation, magnetofection, hydroporation.

[0343] In some embodiments, the vector comprises any of the nucleic acids encoding a CAR described herein. The nucleic acid can be cloned into the vector using any molecular cloning methods known in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. Varieties of promoters have been explored for the expression of genes in mammalian cells and any of the promoters known in the art can be used in the present invention. Promoters can be categorized as constitutive promoters or regulated promoters, such as inducible promoters.

[0344] In some embodiments, the nucleic acid encoding the CAR is operably linked to a constitutive promoter. Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in host cells. Exemplary constitutive promoters contemplated herein include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factors-1alpha (hEF1a), ubiquitin C (UbiC) promoter, phosphoglycerinase (PGK) promoter, virus early promoter simiano 40 (SV40) and chicken β-Actin promoter together with the CMV early enhancer (CAGG). The efficiencies of such constitutive promoters in driving transgene expression have been extensively compared in a large number of studies. For example, Michael C. Milone et al compared the efficiencies of CMV, hEF1α, UbiC, and PGK to drive CAR expression in primary human T cells and concluded that the HEF1α promoter not only induced the highest level of transgene expression, but it was also optimally maintained in human CD4 and CD8 T cells (Molecular Therapy, 17(8):1453-1464 (2009)). In some embodiments, the nucleic acid encoding the CAR is operably linked to a hEF1α promoter.

[0345] In some embodiments, the nucleic acid encoding the CAR is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. The inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the engineered immune effector cell or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducing agent), or a combination thereof. In some embodiments, the induction condition does not induce expression of endogenous genes in the engineered mammalian cell and/or in the subject receiving the pharmaceutical composition. In some embodiments, the induction condition is selected from the group consisting of: inductor, irradiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor environment, and the activation state of the engineered mammalian cell.

[0346] In some embodiments, the vector also contains a selectable marker gene or a reporter gene to select cells that express the CAR from the population of host cells transfected via lentiviral vectors. Both selectable markers and reporter genes can be bound by suitable regulatory sequences to allow expression in host cells. For example, the vector may contain transcription and translation terminators, initiation sequences and promoters useful for regulating expression of the nucleic acid sequences.

[0347] In some embodiments, the vector comprises more than one nucleic acid encoding CARs. In some embodiments, the vector comprises a nucleic acid comprising a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first nucleic acid is operably linked to the second nucleic acid through a third nucleic acid sequence encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, and F2A. In some embodiments, the T2A peptide has an amino acid sequence of SEQ ID NO:385.

Immune effector cells

[0348] "Immune effector cells" are immune cells that can perform immune effector functions. In some embodiments, the immune effector cells express at least FCYRIII and perform ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0349] In some embodiments, the immune effector cells are T cells. In some embodiments, the T cells are CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof. In some embodiments, T cells produce IL-2, TFN, and/or TNF by expressing the CAR and binding to target cells, such as CD20+ or CD19+ tumor cells. In some embodiments, CD8+ T cells lyse antigen-specific target cells by expressing the CAR and binding to the target cells.

[0350] In some embodiments, the immune effector cells are NK cells. In other embodiments, immune effector cells can be established cell lines, e.g., NK-92 cells.

[0351] In some embodiments, immune effector cells are differentiated from a stem cell, such as a hematopoietic stem cell, a pluripotent stem cell, an iPS, or an embryonic stem cell.

[0352] Engineered immune effector cells are prepared by introducing CARs into immune effector cells, such as T cells. In some embodiments, the CAR is introduced into immune effector cells through transfection of any of the isolated nucleic acids or any of the vectors described in Section III. In some embodiments, the CAR is introduced into immune effector cells by inserting proteins into the cell membrane when passing cells through a microfluidic system, such as CELL SQUEEZE® (see, for example, US Patent Application Publication 20140287509).

[0353] Methods of introducing vectors or isolated nucleic acids into a mammalian cell are known in the art. The described vectors can be transferred to an immune effector cell by physical, chemical or biological methods.

[0354] Physical methods for introducing the vector into an immune effector cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cell by electroporation.

[0355] Biological methods for introducing the vector into an immune effector cell include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammals, for example, human cells.

[0356] Chemical means for introducing the vector into an immune effector cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, granules and lipid-based systems, including oil-in-water emulsions, micelles, mixed and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0357] In some embodiments, RNA molecules encoding any of the CARs described herein can be prepared by a conventional method (e.g., in vitro transcription) and then introduced into immune effector cells by known methods, such as mRNA electroporation. . See, for example, Rabinovich et al., Human Gene Therapy 17:1027-1035.

[0358] In some embodiments, the transduced or transfected immune effector cell is propagated ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cell is cultured to propagate for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. . In some embodiments, the transduced or transfected immune effector cell is further evaluated or selected to select the engineered mammalian cell.

[0359] Reporter genes can be used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, for example, enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after the DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase or the green fluorescent protein gene (e.g., Ui-Tei et al. FEBS Letters 479:79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0360] Other methods for confirming the presence of the nucleic acid encoding CARs in the engineered immune effector cell include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR and PCR; biochemical assays, such as detection of the presence or absence of a particular peptide, for example by immunological methods (such as ELISA and Western blots). 1. Sources of T Cells

[0361] Prior to the expansion and genetic modification of T cells, a source of T cells is obtained from an individual. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. . In some embodiments, any number of T cell lines available in the art may be used. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. Again, surprisingly, the initial steps of activation in the absence of calcium lead to enhanced activation. As those skilled in the art would readily appreciate, a washing step can be performed by methods known to those in the art, such as using a "fluid" semiautomatic centrifuge (e.g., the Cobe 2991 Cell Processor, the CytoMate Baxter, or the Haemonetics Cell Saver). 5) in accordance with the manufacturer's instructions. After washing, cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline with or without buffer. Alternatively, undesirable components of the apheresis sample can be removed and the cells are resuspended directly in culture media.

[0362] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ cells, can be additionally isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubating with anti-CD3/anti-CD28 (i.e., 3*28) conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a period of time. sufficient for positive selection of the desired T cells. In some embodiments, the time period is about 30 minutes. In another embodiment, the time period varies from 30 minutes to 36 hours or more and all integer values in between. In yet another embodiment, the period of time is at least 1, 2, 3, 4, 5 or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For isolation of T cells from leukemia patients, using longer incubation periods, such as 24 hours, can increase cell yield. Longer incubation periods can be used to isolate T cells in any situation where there are few T cells compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Furthermore, the use of longer incubation periods may increase the efficiency of CD8+ T cell capture. Thus, by simply reducing or prolonging the time T cells can bind to CD3/CD28 beads and/or by increasing or decreasing the bead to T cell ratio (as described hereinafter), T cell subpopulations can be preferentially selected for or against culture initiation or at other time points during the process. Additionally, by increasing or decreasing the proportion of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, T cell subpopulations can be preferentially selected for or against at culture initiation or at other desired time points. The skilled person would recognize that multiple rounds of selection may also be used. In some embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to more rounds of selection.

[0363] Enrichment of a T cell population by negative selection can be performed with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is the screening and/or selection of cells through negative magnetic immunostaining or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select for regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or another similar selection method.

[0364] For the isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as granules) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which the beads and cells are mixed together (i.e., increase the cell concentration), to ensure maximum cell and bead contact. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In another modality, more than 100 million cells/ml are used. In another embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells/ml is used. In additional embodiments, concentrations of 125 or 150 million cells/ml may be used. The use of high concentrations can result in increased cell yield, cell activation and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or of samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.,). Such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of a high concentration of cells allows for more efficient selection of CD8+ T cells that normally have a lower expression of CD28.

[0365] In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between particles and cells are minimized. This selects cells that express large amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more effective than CD8+ T cells at dilute concentrations. In some embodiments, the cell concentration used is 5*106/mi. In some embodiments, the concentration used can be from approximately 1*105/ml to 1x106/ml, and any integer value in between.

[0366] In some embodiments, cells can be incubated in a rotator for variable long periods at variable speeds at 2-10oC, or at room temperature.

[0367] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freezing and subsequent thawing step provides a more uniform product, removing granulocytes and, to some extent, monocytes in the cell population. After the washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or culture medium containing 10% Dextran 40 and Dextrose. 5%, 20% Human Serum Albumin and 7% DMSO, or 31.25% Plasmalyte-A, 31.25% 5% Dextrose, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, cells are frozen to -80oC at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as uncontrolled freezing immediately at -20oC or in liquid nitrogen.

[0368] In some embodiments, cryopreserved cells are thawed and washed as described herein and allowed to stand for one hour at room temperature before activation.

[0369] Also contemplated in the present application is the collection of blood or apheresis product samples from an individual in a period of time prior to which expanded cells as described herein may be required. As such, the source of cells to be expanded can be harvested at any required time point and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from the therapy. with T cells, such as those described here. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk for developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells can be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein, but prior to any treatment. In a further embodiment, cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunological agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; In another embodiment, cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously, or after) bone marrow or stem cell transplantation, T cell ablative therapy using chemotherapy agents such as fludarabine. , external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated before and can be frozen for later use for treatment after ablative therapy with B cells, such as agents that react with CD20, for example, Rituxan.

[0370] In some embodiments, T cells are obtained from a patient directly after treatment. In this regard, it has been observed that after certain cancer treatments, in specific treatments with drugs that damage the immune system, immediately after treatment during the period in which patients would normally recover from treatment, the quality of the T cells obtained can be optimized or improved its ex vivo expansion capacity. Likewise, following ex vivo manipulation using the methods described here, these cells may be in a preferred state for increased engraftment and expansion in vivo. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage, during this recovery phase. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject in which repopulation, recirculation, regeneration and or expansion of cell types Private therapy is favored, especially during a defined time window after therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

2. T cell activation and expansion

[0371] Whether before or after genetic modification of T cells with the CARs described herein, T cells can be activated and expanded generally using methods as described, for example, in US Patent 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US Patent Application Publication 20060121005.

[0372] Generally, T cells can be expanded by contact with a surface that has attached to it an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein activator. kinase C (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under suitable conditions to stimulate the proliferation of the T cells. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, an antibody anti-CD3 and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR- CD28 (Diaclone, Besancon, France) can be used as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8): 3975-3977, 1998. Haanen et al., J. Exp. Med. 190 (9): 13191328, 1999;

[0373] In some embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activating signal is in solution or coupled to a surface. In certain embodiments, both agents may be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell that expresses Fc receptors or an antibody or other binding agent that binds to the agents. In this regard, see, for example, US Patent Application Publication 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in the activation and amplification of T cells in the present invention.

[0374] In some embodiments, T cells are combined with agent-coated beads, the beads and cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culturing, the agent-coated beads and cells are not separated, but are cultured together. In another embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased binding of cell surface markers, thereby inducing cell stimulation.

[0375] By way of example, cell surface proteins can be bound by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are bound (3 x 28 beads) to contact T cells. , the cells (e.g., 104 to 109 T cells) and beads (e.g., DYNABEADS® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, preferably PBS (without divalent cations, such as calcium and magnesium). Again, those skilled in the art can readily appreciate that any concentration of cells can be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample, or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the scope of the present invention. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase cell concentration), to ensure maximum cell and particle contact. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another modality, more than 100 million cells/ml are used. In another embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells/ml is used. In additional embodiments, concentrations of 125 or 150 million cells/ml may be used. The use of high concentrations can result in increased cell yield, cell activation and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and would be desirable to obtain in certain modalities. For example, the use of a high concentration of cells allows for more efficient selection of CD8+ T cells that normally have a lower expression of CD28.

[0376] In some embodiments, the mixture can be grown for several hours (about 3 hours) to about 14 days or any integer hourly value in between. In another embodiment, the mixture can be cultivated for 21 days. In one embodiment of the invention, the granules and T cells are cultured together for about eight days. In another embodiment, the granules and T cells are cultured together for 2-3 days. Multiple cycles of stimulation may also be desired such that the T cell culture time may be 60 days or more. Appropriate conditions for culturing T cells include an appropriate medium (e.g., Minimum Essential Medium or RPMI 1640 Medium or, X-vivo 15, (Lonza)) that may contain the factors necessary for proliferation and viability, including serum (e.g. example, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other cell growth additives known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactant, plasmanate and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, optimizer, with added amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokines sufficient for T cell growth and expansion. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cell cultures that are to be infused into a subject. Target cells are maintained under conditions necessary to support growth, e.g., an appropriate temperature (e.g., 37 oC) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to varying stimulation times can exhibit different characteristics. For example, typical blood or apheresis peripheral blood mononuclear cell products have a population of T helper cells (TH, CD4+) that is larger than the population of cytotoxic or suppressor T cells (TC, CD8). Ex vivo expansion of T cells through stimulation of CD3 and CD28 receptors produces a population of T cells that, before about days 8-9, consists predominantly of TH cells, whereas after some days 8-9, the T cell population comprises an increasingly large population of TC cells. Consequently, depending on the purpose of the treatment, infusing a subject with a T cell population comprising predominantly TH cells may be advantageous. Likewise, if a specific subset of TC cell antigens is isolated, it may be beneficial to expand this subset to a greater extent.

[0377] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but largely, reproducibly during the cell expansion process. Thus, this reproducibility allows for the ability to tailor an activated T cell product for specific purposes.

V. Pharmaceutical Compositions

[0378] Additionally, there are provided by the present application pharmaceutical compositions comprising any of the anti-BCMA single domain antibodies, or any of the engineered immune effector cells, comprising any of the CARs (such as BCMA CARs) as described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing an sdAb, or a plurality of engineered immune effector cells of the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). , in the form of lyophilized formulations or aqueous solutions.

[0379] Acceptable carriers, excipients or stabilizers are non-toxic to receptors at the dosages and concentrations used and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. protein-Zn complexes); chelating agents, such as EDTA and/or non-ionic surfactants.

[0380] Buffers are used to control pH in a range that optimizes therapeutic efficacy, especially if stability depends on pH. The buffers are preferably present in concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present invention include organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

[0381] Preservatives are added to retard microbial growth and are typically present in a range of 0.2% -1.0% (w/v). Preservatives suitable for use with the present invention include octadecyldimethylbenzylammonium chloride; hexamethylene chloride; benzalkonium halides (e.g. chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0382] Tonicity agents, sometimes known as "stabilizers", are present to adjust or maintain the tonicity of the liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often called "stabilizers" because they can interact with the charged groups of amino acid side chains, thus reducing the potential for inter- and intra-molecular interactions. Polyhydric alcohols may be present in an amount between 0.1% to 25% by weight, typically 1 to 5%, taking into account the relative amounts of the other ingredients. Preferred tonicizing agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0383] Additional excipients include agents that can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adherence to the cell wall. container. Such excipients include: polyhydric sugar alcohols (listed above); amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc. ; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclites (e.g. inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thiotic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0384] Nonionic surfactants or detergents (also known as "wetting agents") are present to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also allows the formulation to be exposed to surface shear stress without causing denaturation of the active therapeutic protein or antibody. Nonionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

[0385] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), polyols from PLURONIC®, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN® -20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0386] For pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition can be made sterile by filtration through sterile filtration membranes. Pharmaceutical compositions of the present document generally are placed in a container having a sterile access port, for example, a vial or bag of intravenous solution that has a stopper pierceable by a hypodermic injection needle.

[0387] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, for example, injection or infusion subcutaneously, intravenously, intraperitoneally, intramuscular, intra-arterial, intralesional or intra-articular, topical administration, inhalation or via sustained release or extended release.

[0388] Extended-release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of molded articles, for example films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Patent 3,773,919), copolymers of L-glutamic acid and ethyl -L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-)-3-hydroxybutyric acid.

[0389] The pharmaceutical compositions described herein may also contain more than one compound or active agent, as necessary, for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or additionally, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0390] The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively, in delivery systems. colloidal drugs (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

SAW. Treatment Methods

[0391] The present application also relates to methods and compositions for use in cellular immunotherapy. In some embodiments, cellular immunotherapy is for the treatment of cancer, including, but not limited to, hematologic malignancies and solid tumors. Any of the anti-BCMA sdAbs, CARs and engineered immune effector cells (such as CAR T cells) described here can be used in the cancer treatment method. The CARs described here may be useful for treating tumors with antigen loss escape mutations and for reducing resistance to existing therapies. In some embodiments, the methods and compositions described herein can be used to treat other diseases that are associated with antigens specifically recognized by single-domain antibodies or CARs, including, for example, autoimmune diseases.

[0392] In some embodiments, there is provided a method of treating a cancer (such as multiple myeloma, e.g., relapsed or refractory multiple myeloma) in an individual (such as a human individual), comprising administering to the individual an effective amount of a composition pharmaceutical comprising:(1) an engineered immune effector cell (such as T cell) comprising a multivalent CAR comprising a polypeptide comprising:(a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety that specifically binds to a first BCMA epitope, and a second BMCA-binding moiety that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier. In some embodiments, there is provided a method of treating a cancer (such as multiple myeloma, e.g., relapsed or refractory multiple myeloma) in a subject (such as a human subject), comprising administering to the subject an effective amount of a pharmaceutical composition comprising: (1) an engineered immune effector cell (such as a T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first anti-BCMA sdAb that specifically binds to a first epitope of BCMA, and a second anti-BMCA sdAb that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier. In some embodiments, the engineered immune effector cell is autologous. In some embodiments, the engineered immune effector cell is allogeneic. In some embodiments, the engineered immune effector cells are CAR T-cells. In some embodiments, the cancer is a liquid cancer, such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is refractory or relapsed multiple myeloma. In some embodiments, the engineered immune effector cell is administered at a dose of about 105 to about 107 cells/kg, such as about 3x105 to about 7x106 cells/kg, or about 3x106 cells/kg. In some embodiments, the engineered immune effector cell is administered by intravenous injection. In some embodiments, the engineered immune effector cell is administered in three divided doses over about a week.

[0393] In some embodiments, there is provided a method of treating a cancer (such as multiple myeloma, e.g., relapsed or refractory multiple myeloma) in an individual (such as a human individual), comprising administering to the individual an effective amount of a composition pharmaceutical comprising: (1) an engineered immune effector cell (such as T cell) comprising a BCMA CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising an anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, and (2) a pharmaceutically acceptable carrier, wherein the anti-BCMA sdAb comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52; and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114. In some embodiments, the extracellular antigen-binding domain comprises at least two anti-BCMA sdAbs. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human or humanized. In some embodiments, the anti-BCMA sdAb comprises a VHH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:115-152. In some embodiments, the BCMA CAR is 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 cell is autologous. In some embodiments, the engineered immune effector cell is allogeneic. In some embodiments, the cancer is a liquid cancer, such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is refractory or relapsed multiple myeloma. In some embodiments, the engineered immune effector cell is administered at a dose of about 105 to about 107 cells/kg, such as about 3x105 to about 7x106 cells/kg, or about 3x106 cells/kg. In some embodiments, the engineered immune effector cell is administered by intravenous injection. In some embodiments, the engineered immune effector cell is administered in three divided doses over about a week.

[0394] In some embodiments, there is provided a method of obtaining partial or complete clinical remission in an individual with multiple myeloma (e.g., relapsed or refractory multiple myeloma), comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) an engineered immune effector cell (such as a T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) that binds specifically to a first BCMA epitope, and a second BCMA binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier. In some embodiments, the engineered immune effector cell is autologous. In some embodiments, the engineered immune effector cell is allogeneic. In some embodiments, the engineered immune effector cells are CAR T-cells. In some embodiments, the cancer is a liquid cancer, such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is refractory or relapsed multiple myeloma. In some embodiments, the engineered immune effector cell is administered at a dose of about 105 to about 107 cells/kg, such as about 3x105 to about 7x106 cells/kg, or about 3x106 cells/kg. In some embodiments, the engineered immune effector cell is administered by intravenous injection. In some embodiments, the engineered immune effector cell is administered in three divided doses over about a week.

[0395] In some embodiments, there is provided a method of treating a cancer (such as multiple myeloma, e.g., relapsed or refractory multiple myeloma) in an individual (such as a human subject), comprising administering to the subject an effective amount of a pharmaceutical composition comprising an anti-BCMA sdAb and a pharmaceutically acceptable carrier, wherein the anti-BCMA sdAb comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52; and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human or humanized. In some embodiments, the anti-BCMA sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NO:115-152. In some embodiments, the cancer is a liquid cancer, such as multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is refractory or relapsed multiple myeloma.

[0396] The methods described here are suitable for treating various types of cancer, including solid cancer and liquid cancer. The methods are applicable to cancers of all stages, including early stage, advanced stage and metastatic cancer. The methods described herein can be used as first therapy, second therapy, third therapy or combination with other types of cancer therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, cell transplantation stem cells, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation or similar, in an adjuvant setting or in a neoadjuvant setting.

[0397] In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is stage I, stage II, or stage III multiple myeloma and/or stage A or stage B based on the Durie-Salmon staging system. In some embodiments, the cancer is stage I, stage II, or stage III multiple myeloma based on the international staging system published by the International Myeloma Working Group (IMWG). In some embodiments, the cancer is monoclonal gammopathy of undetermined significance (MGUS). In some embodiments, the cancer is asymptomatic (smoldering/indolent) myeloma. In some embodiments, the cancer is active or symptomatic myeloma. In some embodiments, the cancer is refractory multiple myeloma. In some embodiments, the cancer is metastatic multiple myeloma. In some embodiments, the individual has not responded to prior treatment for multiple myeloma. In some embodiments, the individual has progressive disease following prior multiple myeloma treatment. In some embodiments, the individual has previously received at least about any of 2, 3, 4 or more treatments for multiple myeloma. In some embodiments, the cancer is relapsed multiple myeloma.

[0398] In some embodiments, the individual has active multiple myeloma. In some embodiments, the individual has at least 10% bone marrow clonal plasma cells. In some embodiments, the individual has a biopsy-proven bone or extramedullary plasmacytoma. In some embodiments, the individual has evidence of end organ damage that can be attributed to the underlying plasma cell proliferative disease. In some embodiments, the individual has hypercalcemia, for example, serum calcium > 0.25 mmol/L (> 1mg/dL) greater than the upper limit of normal or > 2.75 mmol/L (> 11mg/dL). In some embodiments, the individual has renal insufficiency, for example, creatinine clearance < 40 mL per minute or serum creatinine > 177 μmol/L (> 2 mg/dL). In some embodiments, the individual has anemia, for example, a hemoglobin value > 20g/L below the lower limit of normal or a hemoglobin value < 100g/L. In some embodiments, the subject has one or more bone lesions, for example, one or more osteolytic lesions on skeletal radiography, CT, or PET/CT. In some embodiments, the individual has one or more of the following malignancy biomarkers (MDEs): (1) 60% or more clonal plasma cells on bone marrow examination; (2) serum free/uninvolved light chain ratio of 100 or more, provided that the absolute level of involved light chain is at least 100 mg/L; and (3) more than one focal lesion on MRI that is at least 5 mm or more in size.

[0399] Administration of the pharmaceutical compositions can be carried out in any convenient manner, including by injection, ingestion, transfusion, implantation or transplantation. The compositions can be administered to a patient transarterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intraperitoneal. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered to an individual by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676 (1988)). In some embodiments, the pharmaceutical composition is administered to an individual by intradermal or subcutaneous injection. In some embodiments, the compositions are administered by intravenous injection. In some embodiments, the compositions are injected directly into a tumor, or into a lymph node. In one embodiment, the pharmaceutical composition is administered locally to a tumor site, such as directly to tumor cells, or to a tissue that has tumor cells.

[0400] The dosages and desired drug concentration of the pharmaceutical compositions of the present invention may vary depending on the particular intended use. Determination of the appropriate dosage or route of administration is well within the ability of one of ordinary skill. Animal experiments provide reliable guidance for determining effective doses for human therapy. Intraspecies scaling of effective doses can be performed according to the principles established by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may require administration in a manner different from that of another organ or tissue.

[0401] In some embodiments, wherein the pharmaceutical composition comprises any of the sdAbs described herein, the pharmaceutical composition is administered at a dosage of about 10 ng/kg to about 100 mg/kg of the subject's body weight or more per day, for example, at about 1 mg/kg/day to 10 mg/kg/day, depending on the route of administration. Guidance on specific dosages and delivery methods is provided in the literature; see, for example, US Patent 4,657,760; 5, 206, 344; or 5. 225. 212.

[0402] In some embodiments, wherein the pharmaceutical composition comprises any of the engineered immune cells described herein, the pharmaceutical composition is administered at a dosage of at least approximately any one of 104, 105, 106, 107, 108, or 109 cells /kg of the individual's body weight. In some embodiments, the pharmaceutical composition is administered in a dosage of any of about 104 to about 105, about 105 to about 106, about 106 to about 107, about 107 to about 108, about 108 to about 109, about 104 to about 109, about 104 to about 106, about 106 to about 108, or about 105 to about 107 cells/kg of the subject's body weight. In some embodiments, the pharmaceutical composition is administered at a dose of at least about 1x105 2x105 3x105 4x105 5x105 6x105 7x105 8x105 9x105 1x106 2x106 less about x, x, x, x, x, x, x, x, x, x, x , 3x106 4x106 5x106 6x106 7x106 8x106 9x106 1x107 cells/k or x , x , x , x , x , x , x , x c u asg or more. In some embodiments, the pharmaceutical composition is administered at a dose of about 3x10 to about 7x106 cells/kg, or about 3x106 cells/kg.

[0403] In some embodiments, the pharmaceutical composition is administered once. In some embodiments, the pharmaceutical composition is administered multiple times (such as any of 2, 3, 4, 5, 6 or more times). In some embodiments, the pharmaceutical composition is administered once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, or once per year. In some embodiments, the interval between administrations is about 1 week to 2 weeks, 2 weeks to 1 month, 2 weeks to 2 months, 1 month to 2 months, 1 month to 3 months, 3 months to 6 months, or 6 months. months to a year. The optimal dosage and treatment regimen for a particular patient can be easily determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0404] Furthermore, dosages can be administered by one or more separate administrations, or by continuous infusion. In some embodiments, the pharmaceutical composition is administered in separate doses, such as about any one of 2, 3, 4, 5 or more doses. In some embodiments, divided doses are administered over about a week. In some embodiments, the dose is equally divided. In some embodiments, the divided doses are about 20%, about 30%, and about 50% of the total dose. In some embodiments, the interval between consecutive divided doses is about 1 day, 2 days, 3 days or more. For repeated administrations over many days or longer, depending on the condition, treatment is continued until a desired suppression of disease symptoms occurs. However, other dosage regimens may be helpful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0405] In some embodiments, the amount of the pharmaceutical composition is effective to cause an objective clinical response in the individual. In some embodiments, there is provided a method for obtaining an objective clinical response in an individual with multiple myeloma (e.g., relapsed or refractory multiple myeloma), comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) an effector cell engineered immune system (such as T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) that specifically binds to a first BCMA epitope, and a second BCMA binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier. In some embodiments, Strict Clinical Response (sCR) is obtained in the individual.

[0406] In some embodiments, the amount of the pharmaceutical composition is effective to cause remission of the disease (partial or complete) in the individual. In some embodiments, there is provided a method of causing disease remission (partial or complete) in an individual with multiple myeloma (e.g., relapsed or refractory multiple myeloma), comprising administering to the individual an effective amount of a pharmaceutical composition comprising:(1 ) an engineered immune effector cell (such as a T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) which specifically binds to a first BCMA epitope, and a second BCMA-binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier. In some, clinical remission is obtained after no more than any of 6 months, 5 months, 4 months, 3 months, 2 months, 1 month or less after the individual receives the pharmaceutical composition.

[0407] In some embodiments, the amount of the pharmaceutical composition is effective to prevent the recurrence or progression of cancer disease in the individual. In some embodiments, there is provided a method of preventing disease relapse or progression in an individual with multiple myeloma (e.g., relapsed or refractory multiple myeloma), comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) a engineered immune effector cell (such as a T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) that specifically binds to a first BCMA epitope, and a second BCMA binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier. In some embodiments, relapse or progression of the disease is prevented for at least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more.

[0408] In some embodiments, the amount of the pharmaceutical composition is effective to prolong survival (such as disease-free survival) in the individual. In some embodiments, survival is prolonged by at least about 2, 3, 4, 5, 6, 12 or 24 months. In some embodiments, there is provided a method of prolonging the survival of an individual with multiple myeloma (e.g., relapsed or refractory multiple myeloma), comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) an engineered immune effector cell (such as T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) that specifically binds to a first epitope of BCMA, and a second BCMA binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier

[0409] In some embodiments, the pharmaceutical composition is effective in improving the quality of life in the individual. In some embodiments, there is provided a method of improving the quality of life of an individual with multiple myeloma (e.g., relapsed or refractory multiple myeloma), comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) an effector cell engineered immune system (such as T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) that specifically binds to a first BCMA epitope, and a second BCMA binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier

[0410] In some embodiments, the amount of the pharmaceutical composition is effective to inhibit the growth or reduce the size of a solid or lymphatic tumor. In some embodiments, the size of the solid or lymphatic tumor is reduced to at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90% , or 100%). In some embodiments, a method is provided for inhibiting the growth or reducing the size of a solid or lymphatic tumor in an individual.

[0411] In some embodiments, the amount of the pharmaceutical composition is effective to inhibit tumor metastasis in the individual. In some embodiments, at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%) of metastasis is inhibited. In some embodiments, there is provided a method of inhibiting tumor metastasis of an individual with multiple myeloma (e.g., relapsed or refractory multiple myeloma), comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (1) an immune effector cell engineered cell (such as T cell) comprising a multivalent CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising a first BCMA-binding moiety (such as a first anti-BCMA sdAb) that specifically binds to a first BCMA epitope, and a second BCMA binding moiety (such as a second anti-BCMA sdAb) that specifically binds to a second BCMA epitope; (b) a transmembrane domain; and (c) an intracellular signaling domain, in which the first epitope and the second epitope are different; and (2) a pharmaceutically acceptable carrier. In some embodiments, a method of inhibiting lymph node metastasis is provided. In some embodiments, a method of inhibiting metastasis to the lung is provided. In some embodiments, a method of inhibiting metastasis to the liver is provided. Metastasis can be assessed by any methods known in the art, such as blood tests, bone scans, x-ray scans, computed tomography (CT) scans, PET scans and biopsy.

VII. Kits and crafting items

[0412] Kits, unit dosages and articles of manufacture comprising any of the single domain antibodies, chimeric antigen receptors or engineered immune effector cells described herein are also provided. In some embodiments, a kit is provided that contains any of the pharmaceutical compositions described herein and, preferably, provides instructions for their use.

[0413] The kits in this application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) and the like. Kits may optionally provide additional components such as buffers and interpretive information. The present application also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging and the like.

[0414] The article of manufacture may comprise a container and a label or leaflet on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition that is effective for treating a disease or disorder (such as cancer) described herein and may have a sterile access port (e.g., the container may be a bag of intravenous solution or a vial of a piercing cork by a hypodermic injection needle). The label or leaflet indicates that the composition is used to treat the specific condition in an individual. The label or leaflet will also include instructions for administering the composition to the individual. The label may indicate instructions for reconstitution and/or use. The container containing the pharmaceutical composition may be a multiple-use vial, which allows repeated administrations (e.g. 2-6 administrations) of the reconstituted formulation. Insert refers to the instructions usually included in commercial packaging of therapeutic products, which contain information about the indications, use, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container, comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0415] The kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, e.g., hospital pharmacies and compounding pharmacies.

[0416] The examples and exemplary embodiments below are intended to be purely exemplary of the invention and, therefore, should not be considered as limiting the invention in any way. The following exemplary examples and embodiments are offered by way of illustration and not by way of limitation.

VIII. Exemplary modalities

[0417] The invention provides the following modalities:

[0418] Embodiment 1. An anti-BCMA single domain antibody (sdAb), comprising any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO:1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52; and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114.

[0419] Modality 2. The anti-BCMA sdAb of modality 1, wherein the anti-BCMA sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152.

[0420] Modality 3. An anti-BCMA antibody that competes with the anti-BCMA sdAb of modality 1 or 2.

[0421] Modality 4. The anti-BCMA antibody of modality 3, wherein the anti-BCMA antibody is an sdAb.

[0422] Embodiment 5. The anti-BCMA sdAb of any of embodiments 1, 2 and 4, wherein the anti-BCMA sdAb is a camelid antibody.

[0423] Embodiment 6. The anti-BCMA sdAb of any of embodiments 1, 2 and 4, wherein the anti-BCMA sdAb is a chimeric antibody.

[0424] Embodiment 7. The anti-BCMA sdAb of any of modalities 1, 2 and 4, in which the anti-BCMA sdAb is humanized.

[0425] Embodiment 8. The anti-BCMA sdAb of any of embodiments 1-2 and 4-7, wherein the anti-BCMA sdAb is a VHH fragment.

[0426] Embodiment 9. Chimeric antigen receptor (CAR) comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising the anti-BCMA sdAb of any of embodiments 1-2 and 4-7; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0427] Embodiment 10. The CAR of embodiment 9, wherein the extracellular antigen-binding domain comprises at least two anti-BCMA sdAbs.

[0428] Embodiment 11. Multivalent chimeric antigen receptor (CAR), characterized in that it comprises a polypeptide comprising: (a) an extracellular antigen-binding domain comprising at least two BCMA-binding moieties;

(b) a transmembrane domain; and (c) an intracellular signaling domain.

[0429] Embodiment 12. The multivalent CAR of embodiment 11, wherein the extracellular antigen-binding domain comprises a first BCMA-binding moiety and a second BCMA-binding moiety.

[0430] Modality 13. The multivalent CAR of modality 12, wherein one or more of the first BCMA-binding moiety and the second BCMA-binding moiety is an sdAb.

[0431] Modality 14. The multivalent CAR of modality 12 or 13, wherein the first BCMA-binding moiety is a first anti-BCMA sdAb and the second BCMA-binding moiety is a second anti-BCMA sdAb.

[0432] Modality 15. The multivalent CAR of modality 12 or 13, the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is derived from a human antibody.

[0433] Modality 16. The multivalent CAR of modality 12 or 13, wherein the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is a BCMA polypeptide ligand.

[0434] Embodiment 17. The multivalent CAR of any of modalities 12 to 16, wherein the first BCMA-binding moiety and the second BCMA-binding moiety specifically bind to the same epitope on BCMA.

[0435] Embodiment 18. The multivalent CAR of any of embodiments 12-16, wherein the first BCMA-binding moiety and the second BCMA-binding moiety specifically bind to different epitopes on BCMA.

[0436] Embodiment 19. The multivalent CAR of any of embodiments 12 to 18, wherein the first BCMA-binding moiety and/or the second BCMA-binding moiety specifically binds to an epitope on BCMA derived from an amino acid sequence selected from SEQ ID NOs:388-394.

[0437] Modality 20. The multivalent CAR of any of modalities 12 to 19, wherein one or more of the first BCMA-binding moiety and the second BCMA-binding moiety is the anti-BCMA sdAb of modality 1.

[0438] Embodiment 21. The multivalent CAR of any of embodiments 12 to 20, wherein the first BCMA-binding moiety is located at the N-terminus of the second BCMA-binding moiety.

[0439] Embodiment 22. The multivalent CAR of any of embodiments 12 to 20, wherein the first BCMA-binding moiety is located at the C-terminus of the second BCMA-binding moiety.

[0440] Embodiment 23. The multivalent CAR of any of embodiments 12-22, in which the first BCMA-binding moiety and the second BCMA-binding moiety are fused together via a peptide linker.

[0441] Modality 24. The multivalent CAR of modality 23, in which the peptide linker is no more than about 50 amino acids in length.

[0442] Embodiment 25. The multivalent CAR of embodiment 24, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:208-215.

[0443] Embodiment 26. The CAR or multivalent CAR of any one of embodiments 9 to 25, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

[0444] Modality 27. The CAR or multivalent CAR of modality 26, in which the transmembrane domain is derived from CD8α or CD28.

[0445] Modality 28. The CAR or multivalent CAR of modality 27, wherein the transmembrane domain comprises the amino acid sequence of SED ID NO: 193 or 194.

[0446] Embodiment 29. The CAR or multivalent CAR of any one of embodiments 9 to 28, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

[0447] Modality 30. The CAR or multivalent CAR of modality 29, in which the transmembrane domain is derived from CD3Ç

[0448] Modality 31. The CAR or multivalent CAR of modality 30, in which the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 197 or 198.

[0449] Embodiment 32. The CAR or multivalent CAR of any of embodiments 931, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain.

[0450] Modality 33. The CAR or multivalent CAR of modality 32, in which the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA- 1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 Ligands and combinations thereof.

[0451] Modality 34. The CAR or multivalent CAR of modality 33, the co-stimulatory signaling domain comprises a cytoplasmic domain of CD28 and/or a cytoplasmic domain of CD137.

[0452] Modality 35. The CAR or multivalent CAR of modality 34, wherein the co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:195 and/or SEQ ID NO:196.

[0453] Embodiment 36. The CAR or multivalent CAR of any one of embodiments 32 to 35, wherein the intracellular signaling domain comprises at least two co-stimulatory signaling domains.

[0454] Embodiment 37. The CAR or multivalent CAR of any of embodiments 9 to 36, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

[0455] Modality 38. The CAR or multivalent CAR of modality 37, in which the hinge domain is derived from CD8α.

[0456] Embodiment 39. The CAR or multivalent CAR of embodiment 38, wherein the hinge domain comprises the amino acid sequence of SED ID NO:192.

[0457] Embodiment 40. The CAR or multivalent CAR of any one of embodiments 9 to 39, further comprising a signal peptide located at the N-terminus of the polypeptide.

[0458] Modality 41. The CAR or multivalent CAR of modality 40, in which the signal peptide is derived from CD8α.

[0459] Modality 42. The CAR or multivalent CAR of modality 41, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:191.

[0460] Embodiment 43. A chimeric antigen receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 216-256 and 298-335.

[0461] Embodiment 44. An isolated nucleic acid comprising a nucleic acid sequence encoding the CAR or multivalent CAR of any of embodiments 11 to 43.

[0462] Embodiment 45. An isolated nucleic acid of claim 44, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 257-297 and 336-373.

[0463] Embodiment 46. The isolated nucleic acid of embodiment 45, further comprising a second nucleic acid sequence encoding a second CAR, wherein the nucleic acid sequence encoding the CAR is operably linked to the second nucleic acid sequence through a third nucleic acid sequence that encodes a self-cleaving peptide.

[0464] Embodiment 47. The isolated nucleic acid of embodiment 46, the autoclaving peptide is selected from the group consisting of T2A, P2A and F2A.

[0465] Modality 48. The isolated nucleic acid of modality 47, the third nucleic acid sequence is SEQ ID NO:385.

[0466] Embodiment 49. The isolated nucleic acid of any of embodiments 44-48, the isolated nucleic acid is an RNA molecule.

[0467] Embodiment 50. A vector comprising the nucleic acid isolated from any of embodiments 44 to 49.

[0468] Embodiment 51. The vector of embodiment 50, wherein the vector is an expression vector.

[0469] Modality 52. The vector of modality 50 or 51, wherein the vector is a viral vector.

[0470] Modality 53. The vector of modality 52, wherein the vector is a lentiviral vector.

[0471] Modality 54. The vector of modality 50 or 51, wherein the vector is a non-viral vector.

[0472] Embodiment 55. An engineered immune effector cell, comprising the CAR or multivalent CAR of any of modalities 9 to 43, the isolated nucleic acid of any of modalities 44 to 49, or the vector of any of modalities 50 to 54.

[0473] Embodiment 56. The engineered immune effector cell of embodiment 55, wherein the engineered immune effector cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem or an embryonic stem cell.

[0474] Embodiment 57. The engineered immune effector cell of embodiment 56, wherein the immune effector cell is a T cell.

[0475] Embodiment 58. A pharmaceutical composition, comprising the engineered immune effector cell of any one of embodiments 55 to 57 and a pharmaceutically acceptable carrier.

[0476] Modality 59. A method of treating cancer in an individual comprising administering to the individual an effective amount of the pharmaceutical composition of modality 58.

[0477] Modality 60. The method of modality 59, wherein the cancer is multiple myeloma.

[0478] Modality 61. The method of modality 60, in which the cancer is refractory or relapsed multiple myeloma.

[0479] Embodiment 62. The method of any of embodiments 59 to 61, wherein the engineered immune effector cell is autologous.

[0480] Embodiment 63. The method of any of embodiments 59 to 61, wherein the engineered immune effector cell is allogeneic.

[0481] Embodiment 64. The method of any of embodiments 59 to 63, wherein the subject is human.

[0482] Embodiment 65. The method of any one of embodiments 59-64, wherein the pharmaceutical composition is administered intravenously.

[0483] Embodiment 66. The method of any one of embodiments 59 to 65, wherein the pharmaceutical composition is administered at a dose of about 1x105 to about 1x107 cells/kg.

[0484] Embodiment 67. The method of any of embodiments, 59 to 66, in which the pharmaceutical composition is administered in 3 divided doses over about a week.

[0485] Modality 68. Use of the pharmaceutical composition of modality 58 to treat cancer in an individual.

[0486] Modality 69. Use of the pharmaceutical composition of modality 58 in preparing a medicine to treat cancer in an individual.

[0487] Modality 70. A pharmaceutical composition, comprising the anti-BCMA sdAb of any one of modalities 1 to 8.

[0488] Modality 71. A method of treating a disease in an individual comprising administering to the individual an effective amount of the pharmaceutical composition of modality 70.

[0489] Modality 72. The method of modality 71, in which the disease is cancer.

EXAMPLES

[0490] The examples discussed below are intended to be purely exemplary of the invention and should not be considered as limiting the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy regarding the numbers used (e.g. quantities, temperatures, etc.) but some experimental errors and deviations must be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Centigrade, and pressure is atmospheric or near atmospheric.

Example 1. Preparation of anti-BCMA sdAbs

[0491] To develop sdAbs with high binding affinity to BCMA, llamas were immunized with a recombinant BCMA antigen. A phage display library was then constructed to identify VHH leaders. Distinct clones were randomly picked and were classified according to the heavy chain complementarity determining region 3 (CDR3), a region that may play an important role in antigen binding. An exemplary protocol is described below. Other protocols for preparing sdAbs have been described. See, for example, Els Pardon et al, Nature Protocol, 2014; 9(3):674.

1. Animal immunization and immune response assay 1.1 Animal immunization

[0492] An immunogen comprising a recombinant human BCMA protein with a C-terminal Fc tag (ACRO Biosystems, Cat No.: BC7-H5254) was mixed with adjuvant or PBS and injected into llamas. Animals were immunized by the service vendor (Cedarline) seven times, typically with 200 μg of immunogen and CFA (complete Freund's adjuvant) at each interval of approximately 1 week to 2 weeks. Blood samples were collected in the pre-immunization phase and after the 5th and 7th immunization. After several rounds of immunization, the llamas' immune reactions against the target antigen were evaluated to confirm the titer of antigen-specific sdAbs. Lymphocytes were isolated by gradient centrifugation of approximately 100 ml of peripheral blood. Cells were supplemented with RNALATERTM and stored at -80°C. Sera were obtained by centrifugation of anticoagulated blood samples and stored at -80°C.

1.2 IgG fractionation

[0493] IgG subclass fractionation was performed in accordance with the GenScript Standard Operating Procedure. IgG subclasses were fractionated from terminal bleeding serum using Protein G and Protein A proteins. The 1 ml serum sample was loaded onto a 1 ml HITRAP® Protein G HP column, and the column was washed with 10 ml of phosphate buffer (20 mM, pH 7.0). The IgG3 fraction (MW 100,000 Da) was eluted with 0.15 M NaCl, 0.58% acetic acid (pH 3.5), and the eluate was neutralized with 1 M Tris-HCl (pH 9.0) to pH 7 ,4. Subsequently, the IgG1 fraction (MW 170,000 Da) was eluted with 0.1 M glycine-HCl (pH 2.7) and the eluate was neutralized with 1 M Tris-HCl (pH 8.5) until pH 7.4. The flow-through HITRAP® Protein G HP column was then loaded onto a 1 ml HITRAP® Protein A HP column, and the column was washed with 20 ml of phosphate buffer (20 mM, pH 7.0). The IgG2 fraction (MW 100,000 Da) was eluted with 0.15 M NaCl, 0.58% acetic acid (pH 4.5), and the eluate was neutralized with 1 M Tris-HCl (pH 9.0) to pH 7 ,4. The concentrations of purified IgG1, IgG2 and IgG3 antibodies were determined by OD280 and the purity of each was assessed by reducing and non-reducing SDS-PAGE analysis.

1.3 Immune response assay

[0494] The immune response of llamas was assessed by ELISA, in which purified serum and IgG samples were tested for binding to immobilized immunogens. Pre-immunization sera collected, after the 5th immunization and at terminal bleeding were evaluated. Antigen (i.e., recombinant human antigen protein) was diluted in coating buffer to 4 μg/ml. The microtiter plate was coated with diluted antigen at 4°C overnight. The plate was then washed 3 times with wash buffer followed by blocking at room temperature for 2 hours. The plate was subsequently washed 4 times with wash buffer. A series of diluted sera or IgG were added to the plate and incubated at room temperature for 1.5 hours. The plate was then washed 4 times with wash buffer. HRP-conjugated anti-llama IgG secondary antibody was added to the plate and incubated at room temperature for 1 hour. After washing, TMB substrate was added to each well and incubated for 10 minutes before stopping with 1 M HCl. To quantify binding, absorbance at 450 nm was measured for each well using an MK3 spectrometer.

2. Construction of VHH phage display library 2.1 RNA extraction

[0495] Total RNA was extracted from isolated lymphocytes (from 1.1.1) using TRIZOL® Reagent according to the manufacturer's protocol. The quantity and quality of total RNA were assessed by gel electrophoresis and quantified by measuring absorbance at OD260/280. 2.2 RT-PCR and VHH amplification

[0496] Total RNA was reverse transcribed into cDNA with an oligo(dT)20 primer using PRIMESCRIPT™ 1st Strand cDNA Synthesis Kit according to the manufacturer's protocol. Six specific forward and two reverse degenerate primers were designed to amplify the VHH fragments, which had two BglI restriction sites introduced. VHH fragments were amplified according to the GenScript standard operating procedure as described below.

[0497] The variable regions of heavy chain immunoglobulins (i.e., VHHs) were amplified using a two-step polymerase chain reaction (PCR). In the first PCR, 100 ng of cDNA template was mixed with primers CALL001 (SEQ ID NO:374) and CALL002 (SEQ ID NO:375). The DNA products from the first PCR reaction were analyzed by agarose gel electrophoresis. After gel purification, the DNA products from the first PCR were used as templates in the second PCR. The second PCR was performed with the primers BACK-1 (SEQ ID NO:376), BACK-2 (SEQ ID NO:377) and PMCF (SEQ ID NO:378). The second amplified PCR products containing the VHH PCR fragments were gel purified and enzyme digested, and then inserted into phagemid plasmids. The recombinant plasmids with VHH gene fragments were electrotransferred into E. coli cells to generate the VHH phage display immune library.

[0498] The PCR reaction procedure has an initial denaturation step at 94oC for 7 minutes, followed by 30 cycles of 94oC for 1 min, 55oC for 1 min, and 72oC for 1 min; and followed by a final extension step at 72oC for 7 min.

2.3 Phage library construction

[0499] VHH PCR products were obtained by amplification using different pairs of primers. The PCR products were then digested with BglI and gel purified. The gel-purified fragments were inserted into GenScript's in-house phagemid vector. A pilot library was built to optimize the binding and transformation conditions. Optimized ligation and transformation conditions were employed to develop the phagemid library. A small portion of the transformed cells were diluted and streaked onto 2x YT plates supplemented with 100 μg/ml ampicillin. Colonies were counted to calculate library size. Positive clones were randomly selected and sequenced to assess library quality. The rest of the transformed cells were streaked onto YT plates supplemented with 100 μg/ml ampicillin and 2% glucose. Parts of colonies were scraped off the dishes. A small aliquot of the cells was used to isolate the plasmid from the library. The remainder was supplemented with glycerol and stored at −80°C as stock.

3. Panning for phage display 3.1 Biopanning

[0500] The constructed VHH phage library was analyzed against recombinant human BCMA protein and CHO cells expressing human BCMA (i.e., CHO-BCMA cells, prepared in-house by Legend Biotec), respectively, using a standard procedure developed by GenScript. The library stock was grown to logarithmic phase and then the library was rescued with M13KO7 helper phage and was amplified overnight at 25°C on a shaker. The phage was then precipitated with PEG/NaCl, resuspended in PBS, and stored at −80°C. For solid phase panning, microplate wells were coated with recombinant human BCMA protein in PBS at 4°C overnight. For liquid phase filtration, CHO-BCMA cells were blocked with blocking buffer at room temperature for 1 hour. During the coating or blocking step, phage particles were preincubated with blocking buffer and Fc control protein in microplate wells. After pre-incubation, phage particles were added to wells coated with BCMA proteins or CHO-BCMA solution respectively and incubated for 1 hour. After incubation, bound and non-specifically bound phage were washed by washing the wells or CHO-BCMA cells with PBST for six times supplemented with two additional washes of PBS. Bound phage particles were eluted by 100 mM triethylamine (TEA), and the eluate was neutralized by 1 M Tris-HCl (pH 7.4). Half of the eluate was used to infect exponentially growing E. coli TG1 cells (OD600 = 0.4~ 0.6) for output titration.

3.2 Phage ELISA

[0501] Phage ELISA was performed to identify specific clones of the target antigens. Individual output phage clones were grown in 96-deep well plates and rescued by M13KO7 helper phage overnight. To identify clones that bind antigen proteins, 96-well ELISA microtiter plates were coated with recombinant human BCMA protein and Fc control protein, respectively, in coating buffer overnight at 4°C and the plates were then blocked with blocking buffer. After blocking, approximately 50 μl per well of phage supernatant from the overnight cell culture was added to the plates for 1.5 hours of incubation at 4°C. The plates were washed four times and HRP-conjugated anti-M13 monoclonal antibody was added to the plates for 45 minutes of incubation at 4°C. The plates were again washed five times and the substrate solution was added to the wells for development. Absorption at 450 nm was measured for each well.

[0502] To identify clones that bind to CHO-BCMA cells, CHO-BCMA cells were blocked with blocking buffer at room temperature for 1 hour. After blocking, approximately 20 μl per well of overnight cell culture phage supernatant was added to the cell solutions for 1 hour incubation at room temperature. After the cells were washed 4 times, HRP-conjugated anti-M13 monoclonal antibody was added for 30 minutes of incubation at room temperature. Cells were washed five times and substrate solution was then added for development. Absorption was measured at 450 nm. After panning, ELISA-positive phage clones were randomly selected and DNA was prepared from output phage using plasmid extraction kits. The anti-BCMA VHHs on the plasmids were sequenced.

Example 2. Preparation of exemplary monovalent BCMA chimeric antigen receptors

[0503] A nucleic acid sequence encoding a CAR backbone polypeptide comprising from the N-terminus to the C-terminus: a CD8α hinge domain, a CD28 transmembrane domain, a CD28 cytoplasmic domain, a CD137 cytoplasmic domain, and a CD3Z cytoplasmic domain were chemically synthesized and cloned into a premodified downstream lentiviral vector and operably linked to a constitutive hEF1α promoter. The resulting CAR backbone vector was named “PLLV-hEF1α-8281373”. Multicloning sites (MCS) in the vector allowed insertion of a nucleic acid sequence comprising a Kozak sequence (SEQ ID NO:379) operably linked to a nucleic acid sequence encoding a CD8α signal peptide fused to the N-terminus of a VHH fragment in the PLLV-hEF1α-8281373 vector, upstream and operably linked to the CAR backbone sequence.

[0504] To construct a monospecific CAR with a single VHH domain using the PLLV-hEF1α-8281373 backbone, the nucleic acid sequence encoding the VHH domain was operably linked to the 3' of the nucleic acid sequence encoding the signal peptide CD8α. The fusion nucleic acid sequence was chemically synthesized and cloned into the backbone of CAR PLLV-HEF1a-8281373 via EcoRI (SEQ ID NO:380:5'-GAATTC-3') and SpeI (SEQ ID NO: 381: sites of restriction 5'- ACTAGT-3') by molecular cloning techniques known in the art. Table 4 lists the vectors that were constructed to express exemplary monovalent monospecific anti-BCMA CARs.

[0505] To facilitate further insertion of additional sequences, such as a nucleotide encoding a second VHH, when designing a monospecific CAR construct, restriction sites including HpaI (SEQ ID NO:382:5'-GTTAAC-3'), MluI (SEQ ID NO:383:5'-ACGCGT-3'), NsiI (SEQ ID NO:384:5'-ATGCAT-3') sites were included between the nucleic acid sequence of the CD8α signal peptide and the VHH nucleic acid sequence.

[0506] The lentivirus packaging plasmid mixture including pCMV-ΔR- 8.74 and pMD2.G (Addgene #12259) was premixed with the vectors PLLV-hEF1a- 8281373 having VHH fragments in a pre-optimized ratio with polyetherimide (PEI ), then mixed properly and incubated at room temperature for 5 minutes. The transfection mixture was then added dropwise to HEK293 cells and mixed gently. Then, cells were incubated overnight at 37°C and 5% CO2 cell incubator. Supernatants were collected after centrifugation at 4°C, 500 g for 10 min.

[0507] Virus-containing supernatants were filtered through a 0.45 μm PES filter, followed by ultracentrifugation to concentrate lentivirus. After ultracentrifugation, the supernatants were carefully discarded and the virus pellets were cautiously rinsed with pre-chilled DPBS. The virus was aliquoted correctly, then stored at −80°C immediately. Virus titer was determined by p24 based on the HTRF kit developed by GenScript.

PBMC Preparation:

[0508] Leukocytes were collected from healthy donors by apheresis and the cell concentration was adjusted to 5*106 cells/ml in R10 medium. Leukocytes were then mixed with 0.9% NaCl solution at a ratio of 1:1 (v/v). 3 ml lymphoprep medium was added to a 15 ml centrifuge tube and 6 ml of diluted lymphocyte mixture was slowly layered on top of the lymphoprep medium. The lymphocyte mixture was centrifuged at 800 g for 30 minutes without stopping at 20°C. The buffy coat of lymphocytes was then collected with a 200 μl pipette. The collected fraction was diluted at least 6 times with 0.9% NaCl or R10 to reduce the density of the solution. The collected fraction was then centrifuged at 250g for 10 minutes at 20°C. The supernatant was aspirated completely, and 10 ml of R10 was added to the cell pellet to resuspend the cell pellet. The mixture was further centrifuged at 250 g for 10 minutes at 20°C. The supernatant was aspirated again. 2 ml of pre-warmed R10 at 37°C with 100 IU/ml IL-2 were added to the cell pellet and the cell pellet was gently resuspended. Cell number was determined after staining with Trypan Blue, and this PBMC sample was ready for further experiments. T cell purification

[0509] Human T cells were purified from PBMCs using the Miltenyi Pan T Cell Isolation Kit (Cat # 130-096-535), following the manufacturer's protocol as described below. Cell number was first determined and the cell suspension was centrifuged at 300 g for 10 minutes. The supernatant was then aspirated completely, and the cell pellets were resuspended in 40 μl of buffer per 107 total cells. 10 μl of Pan T Cell biotin-antibody cocktail was added per 107 total cells, mixed thoroughly and incubated for about 5 minutes in the refrigerator (2~8°C). 30 μl of buffer was then added per 107 cells. 20 μl of Pan T Cell MicroBead Cocktail was added per 107 cells. The cell suspension mixture was mixed well and incubated for another 10 minutes in the refrigerator (2~8°C). A minimum of 500 μl is required for magnetic separation. For magnetic separation, an LS column was placed in the magnetic field of a suitable MACS separator. The column was prepared by washing with 3 ml of buffer. The cell suspension was then applied to the column, and flow through unlabeled cells were collected, which represented the enriched T cell fractions. Additional T cells were collected by washing the column with 3 ml of buffer and collecting unlabeled cells passing through. These unlabeled cells again represented the enriched T cells, and were combined with the flow-through from the previous step. The pooled enriched T cells were then centrifuged and resuspended in R10 + 100 IU/ml IL-2.

[0510] The prepared T cells were subsequently pre-activated for 48-96 hours with Human T Cell Activation/Expansion Kit (Miltenyi #130-091-441) according to the manufacturer's protocol in which MACSiBead anti- -CD3/CD28 were added at a bead-to-cell ratio of 1:2. In vitro cytotoxicity assay

[0511] Pre-activated T cells were transduced with lentivirus stock in the presence of 7 μg/ml polybrene with centrifugation at 1200 g, 32°C for 1.5 h. The transduced cells were then transferred to the cell culture incubator for transgene expression under appropriate conditions.

[0512] On day 3 or day 7 post-transduction, transduced T cells were harvested and co-incubated with tumor cells at a ratio of effector cells (CAR-T) to target cells of 20:1 for 20 hours. Target cells were human multiple myeloma cell line RPMI8226.Luc, human cell line K562.BCMA.Luc cells that recombinantly express BCMA, K562. CD19. Luc cell line that recombinantly expresses CD19, or cells from the human glioblastoma cell line U87MG.Luc. All cell lines were engineered in-house to express firefly luciferase. To test the cytotoxicity of CAR-T on tumor cells, ONE-GLOTM luminescent luciferase assay reagents (Promega#E6110) were prepared according to the manufacturer's protocol and added to the co-cultured cells to detect the remaining luciferase activity in the well. . Since luciferase is expressed only in target cells, the luciferase activity remaining in the well directly correlates with the number of viable target cells in the well. Maximum luciferase activity was obtained by adding culture media to target cells in the absence of effector cells. Minimal luciferase activity was determined by the addition of Triton X-100 at a final concentration of 1% at the time cytotoxicity assays were initiated. Specific cytotoxicity was calculated by the formula: % Specific Cytotoxicity = 100% * (1- (RLUsample-RLUmin)/(RLUmax-RLUmin)).

[0513] Exemplary monovalent CARs that target BCMA (CD269) were selected and tested in the cytotoxicity assay. As shown in FIG. 1A, among a first group of monovalent BCMA CARs tested, the selected clones exhibited different levels of cytotoxicity against cells of the RPMI8226.Luc multiple myeloma cell line, with more than 60% of VH H-based CAR-Ts showing >50% cytotoxicity against RPMI8226.Luc cells. CAR-T based on clones 269A37346, 269A37348, 269A37353 and 269A37355 were selected for further testing. In particular, CAR-T based on clones 269A37346, 269A37348, 267A37353 and 269A37355 exhibited potent cytotoxicity against the RPMI8226.Luc cell line of multiple myeloma cells with greater than 20% to 30% cell death of RPMI8226.Luc upon treatment with CAR-T compared to untransduced control T cells (UnT). However, such increased cytotoxicity did not occur against human glioblastoma cell line U87MG.Luc cells (see FIG. 1B). No significant cytotoxicity effects against U87MG.Luc were detected by these VHH-based monovalent CAR T-cells compared to UnT controls. The above observation indicated that some of these clones may be target-specific and potent against BCMA-positive cells.

[0514] A second group of exemplary monovalent BCMA CARs was evaluated for in vitro cytotoxicity. GSS005, a CAR comprising an anti-BMCA scFv, served as a positive control. GSI026, a CAR comprising an anti-EGFRvIII scFv, served as a negative control. As shown in FIGS. 2A-2B, the selected clones exhibited different levels of cytotoxicity against RPMI8226.Luc and BCMA multiple myeloma cells overexpressing the K562.BCMA.Luc stable cell line. Neither clone showed potent cytotoxicity against the BCMA-negative cell line K562.CD19.Luc (FIG. 2C). Among these clones, CAR-Ts based on 269B005S, 269B028S, 269B030S, 269B054S, 269B060S, 269B069S, 269B093S, 269B094S, 269B104S, 269B109S, 269B110S and 2 69B129S were more potent according to cytotoxicity data. IFNgamma release

[0515] Additionally, supernatants from in vitro coculture assays were collected to assess CAR-induced cytokine release, e.g., gamma interferon release (i.e. tIFNγ). As shown in FIG. 3, T cells expressing selected monovalent BCMA CARs released high levels of IFNY when cocultured with target cells expressing BCMA K562.BCMA.Luc. Non-specific CAR-Ts, such as GSI026, or non-transferred T cells (UnT) did not induce IFNY release in coculture. Cytokine release data are consistent with in vitro cytotoxicity data,

Example 3. Preparation of exemplary BCMA multivalent chimeric antigen receptors

[0516] Multivalent VHH-based CARs can be constructed by cloning a nucleic acid sequence encoding multiple copies of a VHH, or multiple different VHHs fused together via peptide linkers into a CAR signal domain backbone vector. Examples of multivalent BCMA CAR constructs are shown in Table 5. These constructs were prepared by fusion of 2-3 anti-BCMA VHHs to glycine-serine peptide linkers followed by direct synthesis of this fusion sequence in combination with a nucleic acid sequence of Kozak-CD8α signal peptide, and cloning into the PLLV-hEF1α-81373 CAR backbone through EcoRI and SpeI restriction sites. Monovalent BCMA CAR constructs were also cloned into the same PLLV-hEF1α-81373 CAR backbone to serve as controls (e.g., GSI5011, GSI5019, and GSI5020, Table 4).

[0517] Lentiviral vectors carrying CAR genes were packaged and titrated with protocols as described in Example 2. Using the protocols described in Example 2, human PBMC were prepared from peripheral blood of volunteers for further isolation of primary T cells using Miltenyi PanT cell isolation kits. Purified T cells were pre-activated and expanded using Miltenyi anti-CD3/CD28 microbeads as described in Example 2. Pre-activated T cells were then transduced with lentivirus stock in the presence of 7 μg/ml polybrene by centrifugation at 1200 g, 32°C for 1.5 h. The transduced cells were then transferred to the cell culture incubator for transgene expression under appropriate conditions.

In vitro cytotoxicity assay

[0518] On day 3 post-transduction, transduced T cells were harvested and co-incubated with tumor cells. To assay the cytotoxicity of CAR-T on tumor cells, ONE-GLOTM luminescent luciferase assay reagents were added to the cocultured cells and the specific cytotoxicity for each CAR-T was measured as described in Example 2.

[0519] In a first experiment, monovalent CAR BCMA (GSI5011), bivalent CAR BCMA (GSI5014), and trivalent CAR BCMA (GSI5015) expressing T cells were co-cultured with RPMI8226.Luc cells at an effector to target ratio of 20: 1 for 20 hours. All three CAR constructs comprise anti-BCMA VHH domains from clone 269A37346. As shown in FIG. 4A, the specific percentage of lysis of RPMI8226.Luc cells were 63.25 ± 2.64% by CAR T-cells expressing GSI5011, 61.04 ± 2.75% by CAR T-cells expressing GSI5014, and 59.57 ± 2 .64% by CAR T cells expressing GSI5015, compared to 0.05% ± 2.33% by non-transduced control T cells (UnT). The tested BCMA CARs possessing different antigen-binding modalities had potent antitumor activity against BCMA-positive cells.

[0520] In a second experiment, exemplary bivalent BCMA CARs (GSI5021-GSI5026) having two different BCMA binding moieties 269A37353 and 269A37917 were tested. Engineered T cells expressing each bivalent BCMA CAR were cocultured with RPMI8226.Luc cells at an effector-to-target ratio of 20:1 for 20 hours. Monovalent BCMA CARs, GSI5019 and GSI5020 were also tested for comparison. As shown in FIG. 4B, the specific lysis percentage of RPMI8226.Luc cells were 88.21 ± 1.29% by CAR T-cells expressing GSI5019, 93.84 ± 1.13% by CAR T-cells expressing GSI5020, 71.45 ± 1 .79% by CAR T-cells expressing GSI5021, 99. 80 ± 0. 45% by CAR T-cells expressing GSI5022, 97.46 ± 0.50% by CAR T-cells expressing GSI5023, 81.29 ± 1.27 % by CAR T-cells expressing GSI5024, 93.50 ± 0.47% by CAR T-cells expressing GSI5025, 87.83 ± 0.23% by CAR T-cells expressing GSI5026, respectively, compared to 13.49% ± 1.75% by untransduced control T cells (UnT). Also, as depicted in FIG. 4C, the specific percentage of lysis of BCMA cell line U87MG.Luc was 2.84 ± 7.41% by CAR T-cells expressing GSI5019, 15.50 ± 2.24% by CAR T-cells expressing GSI5020, 6, 74 ± 3.37% by CAR T-cells expressing GSI5021, 8.03 ± 2.36% by CAR T-cells expressing GSI5022, 9.00 ± 1.88% by CAR T-cells expressing GSI5023, 17.03 ± 2.27% by CAR T cells expressing GSI5024, 16.81 ± 1.98% by CAR T cells expressing GSI5025, -11.55 ± 5.43% by CAR T cells expressing GSI5026, compared to 12. 49% ± 3.79% by untransduced control T cells (UnT). The data suggest that bivalent CARs with different antigen-binding modalities possessed potent antitumor activity against BCMA-positive cells but not against BCMA-negative cells.

[0521] In a third experiment, exemplary bivalent BCMA CARs (i.e., BCAR001-BCAR008) having two different BCMA-binding moieties were constructed, and engineered CAR T-cells expressing the bivalent BCMA CARs were prepared from T cells primaries obtained from patient donor R/R MM #13. In-house developed firefly luciferase-expressing cell lines, including RPMI8226 (human multiple myeloma cell line), A549 (human lung cancer cell line), U87-MG (human glioblastoma cell line), and Raji (lymphoma cell line human Burkitt) were used as target cells and co-cultured with each group of transduced T cells side by side (with effector:target cell ratio of 20:1 or 5:1) for 20 hours in a cell incubator at 37oC/5% of CO2, After completion of co-culture, the remaining luciferase activities (relative light unit, RLU) were tested with ONE-GLOTM luminescent luciferase assay kit (Promega) to evaluate the cytotoxicity of each CAR-T. As shown in FIGS. 5A-5E, bivalent BCMA CAR-Ts had dose-dependent cytotoxicity against RPMI8226.Luc, K562.BCMA.Luc, and Raji.Luc cells, but little cytotoxicity against A549.Luc and U87-MG cells. Luc negative for BCMA. Data in FIG. 5F demonstrate that the cytotoxicity of bivalent BCMA CAR T-cells against tumor cells is BCMA-specific, as CAR T-cells were not cytotoxic against K562 cells. C38. Luc that were CD38+/BCMA-. K562.BCMA.Luc treated BCAR001-BCAR008 CAR T-cells showed only limited residual Luciferase activities (i.e., viable cells) compared to UnT-treated target cells (2.88 ± 0.45%, 12.84 ± 1.67%, 2.22 ± 0.56%, 1.77 ± 0.14%, 2.59 ± 0.28%, 6.58 ± 1.19%, 2.47 ± 0.20%, 6.61 ± 1.47 % for BCAR001-BCAR008, respectively, compared to UnT of 100 ± 3.95%, mean ± standard error). These results demonstrate potent cytotoxicity of bivalent BCMA CAR T-cells against K562.BCMA.Luc cells. BCAR001- BCAR008 CAR T-cells did not show significant cytotoxicity against K562 cells. CD38. Luc as no significant decrease in luciferase activity was detected compared to target cells treated with UnT (111.82 ± 5.11%, 111.72 ± 3.43%, 104.74 ± 0.24%, 95 .04 ± 2.70%, 93.93 ± 7.23%, 97.72 ± 1.86%, 111.90 ± 2.01%, 108.33 ± 4.05%, for BCAR001-BCAR008, respectively , compared to UnT of 100 ± 6.58%, mean ± standard error). These data suggest that the cytotoxicity of bivalent BCMA CAR-T is BCMA dependent. IFNgamma release

[0522] Additionally, supernatants from in vitro coculture assays were collected to evaluate CAR-induced cytokine release (e.g., gamma interferon release, JFNy). As shown in FIGS. 6A-6B, IFNY release in the coculture assays was CAR-dependent and BCMA-specific, which is consistent with the in vitro cytotoxicity data (Table 6). TABLE 6. IFN gamma release in bivalent CAR-T BCMA coculture assays Copy numbers of integrated CAR genes

[0523] The copy numbers of integrated CAR genes for each transduced T cell group were determined by a semiquantitative PCR (q-PCR) assay. Briefly, genomic DNA from each CAR-T group was prepared with Gentra Puregene Cell Kit (Qiagen). Genomic DNA concentration was determined by Nanodrop, and the 10ng genomic DNA sample was processed for a standardized q-PCR assay with SYBR Green Realtime PCR Master mix plus (Toyobo) on the ABI #7300 q-PCR instrument using specific primers of CAR (forward primer 137P2F (SEQ ID NO:398:5'-GTCCTTCTCCTGTCACTGGTTAT-3'; and reverse primer 137P2R, SEQ ID NO:399:5'- TCTTCTTCTTCTGGAAATCGGCA-3'). The relative copy number of each gene of integrated CAR was calculated based on a standard curve established using plasmid containing target sequences.

[0524] As shown in Table 7, a high number of copies of the CAR vector was integrated into the T cell genome in each CAR-T preparation. TABLE 7. Genome integration copy numbers. Example 4. Epitope mapping and differential epitope binding of two VHH domains in LCAR-B38M

[0525] The epitopes of the four anti-BCMA VHH domains have been mapped. An exemplary bivalent BCMA CAR containing two different anti-BCMA VHH domains that specifically bind to different BCMA epitopes was constructed. Bivalent CAR/biepitope comprising VHH1 and VHH2, designated CAR LCAR-B38M, is a multivalent BCMA CAR listed in Table 5. Surface Plasmon Resonance (SPR) Assay

[0526] Each of the four exemplary VHH anti-BCMA sequences was cloned into a vector containing a human IgG1 Fc fragment sequence (hIgG1Fc) to facilitate recombinant expression of VHH-hIgG1Fc BCMA. Recombinant proteins were obtained and purified for SPR assays.

[0527] The affinity of each VHH-hIgG1Fc for BCMA was determined by SPR using a BIACORE® 2000 analytical system (GE Healthcare). Briefly, each VHH-hIgG1Fc protein was covalently coupled to a CM5(s) sensor chip using 4 μg/ml VHH-hIgG1Fc. Recombinant BCMA-His protein (ACRO Biosystems, Cat #BCA-H522y) was serially diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.4 ) and injected at a flow rate of 10 μl/min followed by dissociation. Association and dissociation rate constants were determined using the BIACORE® 2000 version 3.0 evaluation software method (Langmuir binding, local fit, 1:1 binding model). The binding affinities of the four VHH-hIgG1Fcs are shown in Table 8. TABLE 8. Binding affinities of VHH-hIgG1Fcs for human BCMA-His protein.

Binding of recombinant VHH-His proteins to target cells

[0528] Recombinant anti-BCMA VHH-His proteins were constructed by fusing the anti-BCMA VHH sequence to a human albumin signal peptide sequence (N'- MKWVTFISLLFLFSSAYS-C'; SEQ ID NO:386) at the N-terminus, and a 6xHis tag (N'- GSGHHHHHH-C'; SEQ ID NO:387) at the C-terminus. The codons were further optimized for optimal expression in mammalian host cells. The obtained nucleotide sequences were then cloned into a mammalian expression vector pTT5 through the 5'-XbaI and 3'-HindIII restriction sites to provide plasmids, pTT5-LAB001 (for VHH1), pTT5-LAB002 (for VHH2) and pTT5 -LAB003 (for VHH1xVHH2).

[0529] In order to obtain recombinant VHH BCMA proteins, HEK293T cells were transiently transfected with the plasmids. Briefly, 5x10 HEK293T cells were seeded in 10 cm cell culture dishes one day before transfection. The next day, cells were transfected with each plasmid using LIPOFECTAMINETM 2000 Reagent (Thermofisher Scientific, Cat. No.: 11668-019) following the manufacturer's manual. Four days after transfection, the supernatant was harvested and antibody expression levels were detected by ELISA using anti-His HRP tag (Biolegend, Cat. No.:652504). The expression levels of LAB001, LAB002 and LAB003 were 109.31 ng/ml, 152.48 ng/ml and 396.62 ng/ml, respectively.

[0530] The binding affinities of VHH-His LAB001, LAB002 and LAB003 anti-BCMA proteins were determined using cell-based assays. Briefly, serially diluted anti-BCMA VHH-His proteins were incubated with 1x10 target cells (either K562.BCMA.Luc or K562.CD38.Luc cells, which were in-house developed cell lines stably expressing BCMA or CD39 respectively) at 4oC for 2 hours. Subsequently, the cells were centrifuged at 300g for 10min and the supernatant was discarded. Cell pellets were resuspended with DPBS. The cell pellets were washed, centrifuged and the supernatant was discarded two more times. Then, the cell pellets were further resuspended with the detection antibody (THETM His-tag Antibody [FITC], GenScript Cat:A01620) containing buffers for 45min at 4 oC for 2 hours. Subsequently, the cells were centrifuged at 300g for 10min and the supernatant was discarded. The cell pellets were washed, centrifuged and the supernatant was discarded two more times. The binding affinities of LAB001, LAB002, and LAB003 to K562.BCMA.Luc or K562 cells. CD38. Luc were determined using an ATTUNE flow cytometer. TM Nxt. The data was fitted by GraphPad PRISMTM version 6.0 using a “One site - Specific binding with Hill slope”.

[0531] As shown in FIG. 7A-7C, LAB001, LAB002, and LAB003 specifically bind to K562.BCMA.Luc cells in a dose-dependent manner. The binding affinities are 0.079 nM, 0.035 nM, and 0.0047 nM, respectively. None of the antibodies showed significant binding to the BCMA-negative cell line K562. CD38. Luc. Furthermore, LAB003 (VHH1xVHH2) showed significantly higher binding affinity (0.0047nM) than LAB001 (VHH1) or LAB002 (VHH2). Epitope binding

[0532] BCMA (NP_001183, UniProt #Q02223) is a transmembrane protein 184 amino acids long. Human BCMA consists of an extracellular domain (ECD, amino residue number 1-54), a transmembrane domain (TM, amino residue number 55-77), and a cytoplasmic domain (CD, amino residue number 78-184). Additionally, sequence analysis suggests that BCMA has no recognizable signal peptide at its N-terminus (Laabi Y et al. (1992) EMBO J 11:3897-3904; Laabi Y et al. (1994) Nucleic Acids Res 22 :1147-1154; Gras M P (1995) Int Immunol 7:1093-1106; Hong-Bing Shu and Holly Johnson (2000): Natl.

[0533] As also illustrated by the UniProt online database (worldwide web. uniprot. org/uniprot/Q02223), 3 disulfide bonds (Cys-Cys) are located in the ECD of BCMA, which are at positions 8 θ 21, 24 θ 37 and 28 θ 41 (Table 9). The secondary structure of the BCMA ECD from N-terminus to C-terminus consists of a beta chain (aa12- 15), a turn (aa16-19), a beta chain (aa20-23), a helix (aa 24 -27), a beta chain (aa30-32), a helix (aa35 - 37), a turn (aa38-40) and a turn (aa42-44). A structure of the BCMA ECD is shown in FIG. 8A, which is replicated from the PDB database structure across the web world. ebi. B.C. uk/pdbe/entry/pdb/2kn1/. TABLE 9. Human BCMA protein sequences.

[0534] BCMA epitope peptides (269EP001-269EP007) were designed as shown in FIG. 8B and TABLE 10, and chemically synthesized and biotinylated at the N-terminus. The binding affinities of VHH1-hIgG1Fc or VHH2-hIgG1Fc were determined using ELISA. Briefly, the peptides described above were coated on MAXISORPTM ELISA plate at 1 μM overnight at 4oC. The next day, plates were washed with PBST (add 0.5% TWEEN-20) twice, followed by blocking plates with 0.5% BSA at room temperature for 1 h. The plates were then washed with PBST twice, followed by the addition of VHH1-hIgG1Fc or VHH2-hIgG1Fc serially diluted to 10 nM in triplicate, then incubated at 4 oC for 2h. Plates were then washed 3 times with cold PBST, after which goat anti-Llama-HRP (1:1500, Bethyl Lab #A160) was added to each well and further incubated at room temperature for 1 h. Plates were then washed 4 times with PBST and TMB substrates were added to each well and incubated at room temperature for 10-30 min. The plates were then read in a TECAN® 10M microplate reader with absorbance at 450nm. TABLE 10. BCMA epitope peptide sequences.

[0535] As shown in FIG. 9A, VHH1 showed strongest binding to peptide 269EP005 followed by peptide 269EP004. However, VHH1 binding to 269EP003 and 269EP006 was relatively weak compared to 269EP005 and 269EP004. VHH1 tends to bind an epitope located on peptide 269EP005 (i.e., amino acid 24-36 of BCMA ECD), which contains the secondary structures of a helix (aa24-27), a beta chain (aa30-32), and a helix (aa35 - 37).

[0536] As shown in FIG. 9B, VHH2 showed strongest binding to peptide 269EP002 followed by peptide 269EP003. However, VHH1 binding to 269EP001 and 269EP004 was relatively weak compared to 269EP002 and 269EP003. While the first beta chain (aa12-15) and the beta chain (aa20-23) of the BCMA ECD are located mainly in the sequence covered by 269EP002 (aa8-21) and 269EP003 (aa11-23), VHH2 tends to bind to an epitope located on the first two beta chains. Competitive binding assay

[0537] Differential epitope binding of VHH1 and VHH2 was further validated by a cell-based competitive binding assay. A stable CHO cell line overexpressing human BCMA (“CHO-BCMA”) was used in the assay.

[0538] Briefly, 0.5 x 106 CHO-BCMA cells were pre-incubated with 12.5 ng/ml of LAB001 (which contains a 6xHis tag at the C-terminus) at 4 oC for 0.5h in duplicate. Then the serially diluted recombinant VHH2-hIgG1Fc antibody was added to each well of the plate and incubated at 4 oC for another 1 h. After incubation, cells were washed with 500μl of DPBS and centrifuged at 300g for 10min. The cell pellets were resuspended with DPBS containing FITC anti-His tag (1:200, GenScript Cat:A16020), then the cells were washed with 500μl of DPBS and centrifuged at 300g for 10min. Cell pellets were resuspended with DPBS, and were then subjected to FACS analysis on an ATTUNETM Nxt flow cytometer. As an assay control, serially diluted VHH2-hIgG1Fc was directly incubated with CHO-BCMA cells without the presence of LAB001 following identical side-by-side procedures as above. Goat anti-human IgG (Fc-specific)-FITC antibody (Sigma Aldrich Cat: F9512) was used to detect binding of VHH2-hIgG1Fc to CHO-BCMA cells. As shown in FIG. 10, VHH2-hIgG1Fc alone binds to CHO-BCMA in a dose-dependent manner. However, VHH2-hIgG1Fc was not able to compete with VHH1-His binding to CHO-BCMA cells, which indicates different binding sites of VHH1 and VHH2 on BCMA. Example 5. In vivo efficacy of CAR-T LCAR-B38M in xenografted mice

[0539] The in vivo antitumor efficacy of LCAR-B38M CAR-T cells was evaluated in the NCG mouse model (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) with a multiple myeloma tumor xenograft. The LCAR-B38M CAR is a bivalent BCMA CAR with two anti-BCMA VHH domains that target different BCMA epitopes.

[0540] The NCG mouse model was created by sequential CRISPR/Cas9 editing of the Prkdc and Il2rg loci in the NOD/Nju mouse, providing a coisogenic mouse to the NOD/Nju. The NOD/Nju mouse carries a mutation in the Sirpa gene (SIRP α) that allows the engraftment of foreign hematopoietic stem cells. Knockdown of Prkdc generates a SCID-like phenotype without the appropriate formation of T cells and B cells. Knockdown of the Il2rg gene further exacerbates the SCID-like phenotype, while additionally resulting in a decrease in NK cell production. Thus, the NCG mouse is a triple immunodeficiency mouse strain that is more immunocompromised than commonly used immunodeficient mouse strains, including SCID and nude mice.

[0541] Prkdc and Il2rg are part of the SCID (severe combined immunodeficiency) family of genes that affect the maturation and formation of T cells, B cells, NK cells and, to a lesser extent, dendritic cells. Prkdc encodes the catalytic subunit of the enzyme DNA-dependent protein kinase, which is required for V(D)J recombination, a process necessary to propagate antibody diversity in maturing T and B cells. Il2rg encodes the common gamma subunit found in IL-2 and multiple IL receptors (IL-4, IL-7, IL-9, IL-15, and IL-21), which are required to induce cytokine-mediated signaling for maturation of immature lymphocytes (e.g., T, B, and NK cells) and other leukocytes.

[0542] LCAR-B38M CAR T-cells were prepared using T cells from various donors to track the source of T cells that produces CAR-T with the highest efficacy of killing RPMI8226.Luc cells in vitro (FIG. 11). Based on the results in FIG. 11, LCAR-B38M T-CAR cells were prepared using T cells from the selected donor for in vivo animal testing. FIG. 12A shows dose-dependent in vitro cytotoxicity of this batch of LCAR-B38M CAR T-cells. To create the tumor xenograft, NCG mice were intravenously injected with RPMI8226.Luc cells. 14 days later, tumor-engrafted mice were treated with LCAR-B38M CAR T cells or untransduced T cells, followed by in vivo bioluminescence imaging (BLI).

[0543] As shown in FIGS. 12B-12C, LCAR-B38M CAR T-cells were efficient to eradicate RPMI8226.Luc tumor cells engrafted into NCG mice and rescue the mice, while the majority of mice in the control group (UnT) died within 4 weeks. Interestingly, during autopsy, numerous metastatic tumors were observed in the livers of all mice in the UnT group. This observation was further validated by evaluating ex vivo luciferase activities of the tumor samples (FIGS. 12D-12E). In contrast, mice treated with CAR-T LCAR-B38M did not have metastatic tumors in their livers. In summary, the in vivo study demonstrates the potency of LCAR-B38M T-CAR cells in eradicating multiple myeloma cells, e.g., RPMI8226.Luc) from NCG mice.

Example 6. Safety Study of CAR-T LCAR-B38M Treatment in Non-Human Primates

[0544] The in vivo safety of LCAR-B38M T-CAR cells was evaluated in a cynomolgus monkey model. PBMC was obtained from peripheral blood samples from two monkeys (NHP #120117 and NHP #120545, both males, approximately 4 kg) and prepared by density gradient centrifugation. Cynomolgus monkey T cells were isolated from PBMC using non-human primate Pan T Cell Isolation Kit (Miltenyi#130-091-993) according to the manufacturer's manual. Prepared monkey T cells were preactivated with the Non-Human Primate T Cell Activation/Expansion Kit (Miltenyi #130-092-919), human IL-2, and autologous monkey serum for 3 days. Subsequently, pre-activated T cells were transduced with the LCAR-B38M lentivirus, followed by expansion for an additional 10 days.

[0545] 3 days before the infusion of autologous CAR-T cells, the monkeys were pre-treated with Cyclophosphamide at a dose of 22mg/kg of body weight by intravenous infusion. On the day of autologous infusion, cells were thawed in a 37°C water bath by gentle shaking and immediately infused intravenously into the animals within 5 minutes. Monkey NHP #120117 was infused with 5 x 106/kg CAR T-cells and monkey NHP #120545 was infused with 4 x 107/kg CAR T-cells.

[0546] Monkeys were monitored after administration of T cells for fever, respiratory distress, change in appetite, diarrhea and weight loss. Pre- and post-administration blood samples were obtained and examined for blood counts, serum levels, and cytokines. As shown in FIGS. 13A-13F, CAR T cells did not show significant toxicity in monkeys. Example 7. A Clinical Study of CAR-T LCAR-B38M in Human Patients With Relapsed/Refractory Multiple Myeloma

[0547] A phase 1/2, open-label, multicenter, single-arm clinical study was conducted to determine the safety and efficacy of LCAR-B38M CAR T-cells in the treatment of human patients diagnosed with relapsed or refractory multiple myeloma ( “r/r MM”). Information about the clinical trial can be found at worldwide web.clinicaltrials.gov, with the identifier NCT03090659.

[0548] In the study, refractory/relapsed patients with multiple myeloma were treated with LCAR-B38M CAR T-cells derived from the patients' autologous T cells. A total dose of 0.5 x 106 -5 x 106 cells/kg body weight was administered to each patient by intravenous injection in three divided doses (20%, 30% and 50%, respectively) for the duration of one week (for example, on days 0, 2 and 6). During days 1-30 of the study, patients were monitored for adverse events, and patient samples were obtained for laboratory evaluation. All patients are followed for at least 36 months after CAR-T administration.

[0549] The primary outcome of the study measures the occurrence of treatment-related adverse events as assessed by the Common Terminology Criteria for Adverse Events (CTCAE) v4. 0 within 1-30 days after injection of LCAR-B38M CAR T cells. The secondary outcome evaluates CAR-T-induced antimyeloma responses, for example, by determining aberrant serum immunoglobulin levels and multiple myeloma cell numbers in patients' bone marrow before and after administration of LCAR-B38M CAR T-cells. Study efficacy endpoints include pathologic complete response proportion, 3-year disease-free survival, 3-year progression-free survival.

[0550] Patients between the ages of 18 and 75 are eligible for the study if: (1) the patient has a confirmed prior diagnosis of active multiple myeloma as defined by the updated IMWG criteria; (2) Clear BCMA expression is detected in malignant plasma cells from bone marrow or a plasmacytoma by flow cytometry or immunohistochemistry; and (3) the patient has refractory multiple myeloma, as defined by having received at least three prior treatment regimens, including bortezomib, or otherwise identified by clinical physicians; or the patient has relapsed multiple myeloma as defined in the NCCN clinical practice guidelines in Oncology:Multiple Myeloma (2016 V2).

[0551] The following patients are excluded from the study: (1) women of childbearing age or who are pregnant or breastfeeding; (2) patients who have an active and uncontrolled infection: hepatitis B, hepatitis C, HIV or other fatal viral and bacterial infections; (3) patients who received systemic corticosteroid steroid therapy greater than 5 mg/day of prednisone or equivalent dose of another corticosteroid are not allowed within 2 weeks before the required leukapheresis or the start of the conditioning chemotherapy regimen; (4) patients with an uncontrolled intercurrent illness or serious and uncontrolled medical disorder; (5) patients with CNS metastases or symptomatic CNS involvement (including cranial neuropathies or mass lesions and spinal cord compression); (6) patients with a history of allogeneic stem cell transplantation, who have active acute or chronic graft-versus-host disease (GVHD), or require immunosuppressive medications for GVHD, within 6 months of enrollment; or (7) patients with active autoimmune skin diseases such as psoriasis or other active autoimmune diseases such as rheumatoid arthritis.

[0552] In an interim analysis in May 2017, 35 patients with relapsed or treatment-resistant (refractory) multiple myeloma received treatment with CAR-T LCAR-B38M. The first signs of treatment effectiveness appeared 10 days after the initial injection of CAR T-cells. Overall, the objective response rate was 100% and 33 of 35 (94%) patients had an evident clinical remission of myeloma (complete response or very good partial response) within two months of receiving CAR T-cells.

[0553] At the time of analysis, 19 patients had been followed for more than four months, a pre-established time for complete evaluation of efficacy according to the International Myeloma Working Group (IMWG) consensus criteria. One patient achieved partial response and four patients achieved very good partial remission (VgPR) efficacy criteria. No patient who met stringent complete response ("sCR") criteria relapsed. Five patients who were followed for more than a year (12-14 months) remained in CRS status and were free of minimal residual disease (i.e., no detectable cancer cells in the bone marrow).

[0554] Cytokine release syndrome (CRS) is a common and potentially dangerous side effect of CAR T cell therapy. Only transient CRS was experienced in 85% of the 35 patients. Symptoms of CRS include fever, low blood pressure, difficulty breathing, and problems with multiple organs. In most patients, CRS symptoms were mild and manageable. Only two patients experienced severe CRS (grade 3) but recovered upon receiving tocilizumab (an inflammation-reducing treatment commonly used for the management of CRS in CAR T-cell therapy clinical trials). No patients experienced neurological side effects, another common and serious complication of CAR T-cell therapy.

[0555] Interim clinical trial data demonstrate the potent efficacy and safety of LCAR-B38M CAR-T treatment in patients with refractory/relapsed multiple myeloma.

[0556] In a pilot clinical study, 3 patients were treated with autologous T cells expressing a monovalent CAR BCMA, i.e., LCAR-B27S T-CAR cells. The LCAR-B27S CAR (a monovalent BCMA CAR listed in Table 4) has an antigen-binding domain containing a single VHH fragment that recognizes a single epitope of the BCMA molecule. This VHH domain is identical to the second VHH domain of the biepitope/bivalent CAR LCAR-B38M.

[0557] In an in vitro cytotoxicity assay, LCAR-B27S CAR T-cells were prepared from three patients with multiple myeloma, respectively, and LCAR-B38M CAR T-cells were also prepared respectively from the same three patients. with multiple myeloma as control. Both CAR T-cells were co-cultured with RPMI8226.Luc cells at an effector-to-target ratio (E/T ratio) of 20:1 and 5:1 for 20 hours. As shown in FIG. 14A, CAR T cells were prepared using T cells from patient A. The percentage of remaining viable cells, assessed by remaining luciferase activity in RPMI8226.Luc cells, was 3.97 ± 0.75% for LCAR-B38M and 3.17 ± 0.57% for LCAR-B27S, when the E/T ratio was 20:1. However, when the E/T ratio was 5:1, LCAR-B38M showed higher killing potencies of RPMI8226.Luc cells compared to LCAR-B27S (33.37 ± 0.75% viable cells remaining for LCAR- B38M, 68.60 ± 1.60% for LCAR-B27S). As shown in FIG. 14B, CAR-T cells were prepared using T cells from patient B. The percentage of remaining viable cells, assessed by remaining luciferase activity in RPMI8226.Luc cells, was 4.45 ± 0.57% for LCAR-B38M and 9.32 ± 1.16% for LCAR-B27S, when the E/T ratio was 20:1. However, when the E/T ratio was 5:1, LCAR-B38M again showed higher killing potencies of RPMI8226.Luc cells compared to LCAR-B27S (40.92 ± 3.00% viable cells remaining for LCAR -B38M, 84.05 ± 1.56% for LCAR-B27S). As shown in FIG. 14C, CAR T-cells were prepared using T cells from patient C. The percentage of remaining viable cells, assessed by remaining luciferase activity in RPMI8226.Luc cells, was 2.56 ± 0.88% for LCAR-B38M and 10 .12 ± 1.83% for LCAR-B27S, when the E/T ratio was 20:1. However, when the E/T ratio was 5:1, LCAR-B38M again showed higher RPMI8226.Luc cell killing potencies compared to LCAR-B27S (29.99 ± 3.13% viable cells remaining for LCAR- B38M, 100.93 ± 9.25% for LCAR -B27S).

[0558] In the pilot clinical study, 3 patients were treated with the LCAR-B27S CAR T-cells, in which all preconditioning, injection and follow-up protocols were identical to those in the clinical study with the bivalent CAR LCAR-B38M. A total dose of 3 x 106 cells/kg (patient A), 5 x 106 cells/kg (patient B) and 7 x 106 cells/kg (patient C) per body weight of autologous LCAR-B27S modified CAR T cells were administered to each patient respectively by intravenous injection in three divided doses (20%, 30% and 50% respectively) over the course of one week (e.g. on days 0, 2 and 6). During days 1-30 of the study, patients were monitored for adverse events, and patient samples were obtained for laboratory evaluation. All patients were followed for at least 36 months after CAR-T administration.

[0559] 2 patients among the three patients achieved a very good partial response (VgPR), but both patients relapsed within 6 months after CAR-T infusion. The third patient did not show a clinical response. The pilot study with LCAR-B27S was consequently terminated by the IRB without further patient enrollment.

[0560] The objective response rate, complete remission rate, and relapse rate of this pilot monovalent CAR BCMA study (LCAR-B27S) and the bivalent CAR BCMA study (CAR-T LCAR-B38M) are shown in the Table 11 below. Bivalent CAR-T BCMA showed superior clinical efficacy compared to monovalent CAR-T BCMA. TABLE 11: Comparable clinical data from monovalent and bivalent/biepitope CAR-T BCM therapies

Claims (43)

1. Multivalent chimeric antigen receptor (CAR), characterized in that it comprises a polypeptide comprising: (a) an extracellular antigen-binding domain comprising at least two BCMA-binding moieties; (b) a transmembrane domain; and (c) an intracellular signaling domain. 2. Multivalent CAR according to claim 1, characterized in that the extracellular antigen-binding domain comprises a first anti-BCMA binding moiety and a second BCMA-binding moiety. 3. Multivalent CAR according to claim 1 or 2, characterized in that one or more of the first BCMA-binding moiety and the second BCMA-binding moiety is an anti-BCMA sdAb. 4. Multivalent CAR according to claim 2 or 3, characterized in that the first BCMA-binding moiety is a first anti-BCMA sdAb and the second BCMA-binding moiety is a second anti-BCMA sdAb. 5. Multivalent CAR according to claim 2 or 3, characterized in that the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is derived from a human antibody. 6. Multivalent CAR according to claim 2 or 3, characterized in that the first BCMA-binding moiety is an anti-BCMA sdAb and the second BCMA-binding moiety is a BCMA polypeptide ligand. 7. Multivalent CAR according to any one of claims 2 to 6, characterized in that the first BCMA-binding moiety and the second BCMA-binding moiety specifically bind to the same epitope on BCMA. 8. Multivalent CAR according to any one of claims 2 to 6, characterized in that the first BCMA-binding moiety and the second BCMA-binding moiety specifically bind to different epitopes in BCMA. 9. Multivalent CAR according to any one of claims 2 to 8, characterized in that the first BCMA-binding moiety and/or the second BCMA-binding moiety specifically binds to an epitope on BCMA derived from a amino acid sequence selected from SEQ ID NOs:388-394. 10. Multivalent CAR according to any one of claims 3 to 9, characterized in that the anti-BCMA sdAb comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52 and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114. 11. Multivalent CAR according to any one of claims 2 to 10, characterized in that the first BCMA-binding moiety is located at the N-terminus of the second BCMA-binding moiety. 12. Multivalent CAR according to any one of claims 2 to 10, characterized in that the first BCMA-binding moiety is located at the C-terminus of the second BCMA-binding moiety. 13. Multivalent CAR according to any one of claims 2 to 12, characterized in that the first BCMA-binding moiety and the second BCMA-binding moiety are fused together via a peptide linker. 14. Multivalent CAR according to claim 13, characterized in that the peptide linker is no more than about 50 amino acids in length. 15. Anti-BCMA single domain antibody (sdAb), characterized by the fact that it comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO:39; and a CDR3 comprising the amino acid sequence of SEQ ID NO:77; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO:2; a CDR2 comprising the amino acid sequence of SEQ ID NO:40; and a CDR3 comprising the amino acid sequence of SEQ ID NO:78; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO:3; a CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a CDR3 comprising the amino acid sequence of SEQ ID NO:79; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:4; a CDR2 comprising the amino acid sequence of SEQ ID NO:42; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:5; a CDR2 comprising the amino acid sequence of SEQ ID NO:43; and a CDR3 comprising the amino acid sequence of SEQ ID NO:81; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:6; a CDR2 comprising the amino acid sequence of SEQ ID NO:44; and a CDR3 comprising the amino acid sequence of SEQ ID NO:82; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:45; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO:8; a CDR2 comprising the amino acid sequence of SEQ ID NO:46; and a CDR3 comprising the amino acid sequence of SEQ ID NO:84; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO:9; a CDR2 comprising the amino acid sequence of SEQ ID NO:47; and a CDR3 comprising the amino acid sequence of SEQ ID NO:85; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO:10; a CDR2 comprising the amino acid sequence of SEQ ID NO:48; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO:11; a CDR2 comprising the amino acid sequence of SEQ ID NO:49; and a CDR3 comprising the amino acid sequence of SEQ ID NO:87; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO:12; a CDR2 comprising the amino acid sequence of SEQ ID NO:50; and a CDR3 comprising the amino acid sequence of SEQ ID NO:88; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO:13; a CDR2 comprising the amino acid sequence of SEQ ID NO:51; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89; (14) a CDR1 comprising the amino acid sequence of SEQ ID NO:14; a CDR2 comprising the amino acid sequence of SEQ ID NO:52 and a CDR3 comprising the amino acid sequence of SEQ ID NO:90; (15) a CDR1 comprising the amino acid sequence of SEQ ID NO:15; a CDR2 comprising the amino acid sequence of SEQ ID NO:53; and a CDR3 comprising the amino acid sequence of SEQ ID NO:91; (16) a CDR1 comprising the amino acid sequence of SEQ ID NO:16; a CDR2 comprising the amino acid sequence of SEQ ID NO:54; and a CDR3 comprising the amino acid sequence of SEQ ID NO:92; (17) a CDR1 comprising the amino acid sequence of SEQ ID NO:17; a CDR2 comprising the amino acid sequence of SEQ ID NO:55; and a CDR3 comprising the amino acid sequence of SEQ ID NO:93; (18) a CDR1 comprising the amino acid sequence of SEQ ID NO:18; a CDR2 comprising the amino acid sequence of SEQ ID NO:56; and a CDR3 comprising the amino acid sequence of SEQ ID NO:94; (19) a CDR1 comprising the amino acid sequence of SEQ ID NO:19; a CDR2 comprising the amino acid sequence of SEQ ID NO:57; and a CDR3 comprising the amino acid sequence of SEQ ID NO:95; (20) a CDR1 comprising the amino acid sequence of SEQ ID NO:20; a CDR2 comprising the amino acid sequence of SEQ ID NO:58; and a CDR3 comprising the amino acid sequence of SEQ ID NO:96; (21) a CDR1 comprising the amino acid sequence of SEQ ID NO:21; a CDR2 comprising the amino acid sequence of SEQ ID NO:59; and a CDR3 comprising the amino acid sequence of SEQ ID NO:97; (22) a CDR1 comprising the amino acid sequence of SEQ ID NO:22; a CDR2 comprising the amino acid sequence of SEQ ID NO:60; and a CDR3 comprising the amino acid sequence of SEQ ID NO:98; (23) a CDR1 comprising the amino acid sequence of SEQ ID NO:23; a CDR2 comprising the amino acid sequence of SEQ ID NO:61; and a CDR3 comprising the amino acid sequence of SEQ ID NO:99; (24) a CDR1 comprising the amino acid sequence of SEQ ID NO:24; a CDR2 comprising the amino acid sequence of SEQ ID NO:62; and a CDR3 comprising the amino acid sequence of SEQ ID NO:100; (25) a CDR1 comprising the amino acid sequence of SEQ ID NO:25; a CDR2 comprising the amino acid sequence of SEQ ID NO:63; and a CDR3 comprising the amino acid sequence of SEQ ID NO:101; (26) a CDR1 comprising the amino acid sequence of SEQ ID NO:26; a CDR2 comprising the amino acid sequence of SEQ ID NO:64; and a CDR3 comprising the amino acid sequence of SEQ ID NO:102; (27) a CDR1 comprising the amino acid sequence of SEQ ID NO:27; a CDR2 comprising the amino acid sequence of SEQ ID NO:65; and a CDR3 comprising the amino acid sequence of SEQ ID NO:103; (28) a CDR1 comprising the amino acid sequence of SEQ ID NO:28; a CDR2 comprising the amino acid sequence of SEQ ID NO:66; and a CDR3 comprising the amino acid sequence of SEQ ID NO:104; (29) a CDR1 comprising the amino acid sequence of SEQ ID NO:29; a CDR2 comprising the amino acid sequence of SEQ ID NO:67; and a CDR3 comprising the amino acid sequence of SEQ ID NO:105; (30) a CDR1 comprising the amino acid sequence of SEQ ID NO:30; a CDR2 comprising the amino acid sequence of SEQ ID NO:68; and a CDR3 comprising the amino acid sequence of SEQ ID NO:106; (31) a CDR1 comprising the amino acid sequence of SEQ ID NO:31; a CDR2 comprising the amino acid sequence of SEQ ID NO:69; and a CDR3 comprising the amino acid sequence of SEQ ID NO:107; (32) a CDR1 comprising the amino acid sequence of SEQ ID NO:32; a CDR2 comprising the amino acid sequence of SEQ ID NO:70; and a CDR3 comprising the amino acid sequence of SEQ ID NO:108; (33) a CDR1 comprising the amino acid sequence of SEQ ID NO:33; a CDR2 comprising the amino acid sequence of SEQ ID NO:71; and a CDR3 comprising the amino acid sequence of SEQ ID NO:109; (34) a CDR1 comprising the amino acid sequence of SEQ ID NO:34; a CDR2 comprising the amino acid sequence of SEQ ID NO:72; and a CDR3 comprising the amino acid sequence of SEQ ID NO:110; (35) a CDR1 comprising the amino acid sequence of SEQ ID NO:35; a CDR2 comprising the amino acid sequence of SEQ ID NO:73; and a CDR3 comprising the amino acid sequence of SEQ ID NO:111; (36) a CDR1 comprising the amino acid sequence of SEQ ID NO:36; a CDR2 comprising the amino acid sequence of SEQ ID NO:74; and a CDR3 comprising the amino acid sequence of SEQ ID NO:112; (37) a CDR1 comprising the amino acid sequence of SEQ ID NO:37; a CDR2 comprising the amino acid sequence of SEQ ID NO:75; and a CDR3 comprising the amino acid sequence of SEQ ID NO:113; or (38) a CDR1 comprising the amino acid sequence of SEQ ID NO:38; a CDR2 comprising the amino acid sequence of SEQ ID NO:76; and a CDR3 comprising the amino acid sequence of SEQ ID NO:114. 16. anti-BCMA sdAb according to claim 15, characterized in that the anti-BCMA sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 115-152. 17. Anti-BCMA antibody, characterized by the fact that it competes with the anti-BCMA sdAb as defined in claim 15 or 16. 18. Anti-BCMA antibody according to claim 17, characterized in that the anti-BCMA antibody is an sdAb. 19. anti-BCMA sdAb according to any one of claims 15, 16 and 18, characterized in that the anti-BCMA sdAb is a camelid antibody. 20. anti-BCMA sdAb according to any one of claims 15, 16 and 18, characterized in that the anti-BCMA sdAb is a chimeric antibody. 21. anti-BCMA sdAb according to any one of claims 15, 16 and 18 to 20, characterized in that the anti-BCMA sdAb is a VHH fragment. 22. A chimeric antigen receptor (CAR) comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising the anti-BCMA sdAb of any one of claims 15 to 16 and 18 to 21; (b) a transmembrane domain; and (c) an intracellular signaling domain. 23. CAR, according to claim 22, characterized by the fact that the extracellular antigen binding domain comprises at least two anti-BCMA sdAbs. 24. CAR or multivalent CAR according to any one of claims 1 to 14 and 23 to 23, characterized in that the transmembrane domain is derived from a molecule selected from the group consisting of CD8a, CD4, CD28, CD137, CD80 , CD86, CD152 and PD1. 25. CAR or multivalent CAR according to claim 24, characterized in that the transmembrane domain is derived from CD8α or CD28. 26. CAR or multivalent CAR according to any one of claims 1 to 14 and 22 to 25, characterized in that the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. 27. CAR or multivalent CAR according to claim 26, characterized in that the transmembrane domain is derived from CD3α. 28. CAR or multivalent CAR according to any one of claims 1 to 14 and 22 to 27, characterized in that the intracellular signaling domain comprises a co-stimulatory signaling domain. 29. CAR or multivalent CAR according to claim 28, characterized in that the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 Ligands and combinations thereof. 30. CAR or multivalent CAR according to claim 29, characterized in that the co-stimulatory signaling domain comprises a cytoplasmic domain of CD28 and/or a cytoplasmic domain of CD137. 31. CAR or multivalent CAR according to any one of claims 1 to 14 and 22 to 30, characterized in that it further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N- terminal of the transmembrane domain. 32. CAR or multivalent CAR, according to claim 31, characterized by the fact that the transmembrane domain is derived from CD8α. 33. CAR or multivalent CAR, according to any one of claims 1 to 14 and 22 to 32, characterized in that it further comprises a signal peptide located at the N-terminus of the polypeptide. 34. CAR or multivalent CAR according to claim 33, characterized in that the signal peptide is derived from CD8α. 35. Chimeric antigen receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 216-256 and 298-335. 36. Isolated nucleic acid, characterized by the fact that it comprises a nucleic acid sequence that encodes the CAR or multivalent CAR as defined in any of embodiments 1 to 14 and 22 to 35. 37. Vector, characterized by the fact that it comprises the nucleic acid as defined in claim 36. 38. Engineered immune effector cell, characterized by the fact that it comprises the CAR or multivalent CAR as defined in any one of claims 1 to 14 and 22 to 35, the isolated nucleic acid as defined in claim 36 or the vector as defined in claim 37 . 39. The engineered immune effector cell of claim 38, wherein the immune effector cell is a T cell. 40. Pharmaceutical composition, characterized by the fact that it comprises the engineered immune effector cell of claim 39 or the BCMA sdAb of any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 41. Method of treating cancer in an individual, characterized by the fact that it comprises administering to the individual an effective amount of the pharmaceutical composition as defined in claim 40. 42. Method according to claim 41, characterized by the fact that the cancer is multiple myeloma. 43. Method according to claim 42, characterized by the fact that the cancer is refractory or relapsed multiple myeloma.