Single-domain antibodies and chimeric antigen receptors targeting bcma and methods of use thereof

Abstract

A chimeric antigen receptor (CAR) comprising a polypeptide comprising an extracellular antigen binding domain comprising a first BCMA binding moiety and a second BCMA binding moiety, wherein the first BCMA binding moiety is a first anti-BCMA single domain antibody, and the second BCMA binding moiety is a second anti-BCMA sdAb; and wherein each of the first sdAb and the second sdAb is a VHH domain.

Description

[0001] Cross-Reference to Related Applications

[0002] This application claims priority to International Patent Application No. PCT / CN2019 / 125681, filed December 16, 2019, International Patent Application No. PCT / CN2020 / 112181, filed August 28, 2020, and International Patent Application No. PCT / CN2020 / 112182, filed August 28, 2020, the contents of each of which are incorporated by reference herein in their entirety.

[0003] Sequence Listing

[0004] The application incorporates by reference the sequence listing, which is submitted in text format with the application, entitled “14651-013-228_SEQ_LISTING,” created on December 14, 2020, having a size of 118,439 bytes. 1. TECHNICAL FIELD

[0005] Provided are single domain antibodies targeting BCMA, and chimeric antigen receptors (e.g., multivalent CARs, including bi-epitope CARs) comprising one or more anti-BCMA single domain antibodies. Also provided are engineered immune effector cells (e.g., T cells) comprising the chimeric antigen receptors. Also provided are pharmaceutical compositions, kits, and methods of treating cancer. 2. BACKGROUND

[0006] B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is preferentially expressed by mature B-lymphocytes, and its overexpression and activation are associated with human cancers such as multiple myeloma. Shah et al., Leukemia, 34:985-1005 (2020).

[0007] Multiple myeloma (MM) is an incurable, aggressive plasma cell malignancy classified as a B-cell neoplasm that proliferates uncontrollably in the bone marrow, interfering with the normal 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 insufficiency, anemia, bone lesions, bacterial infections, hyperviscosity, and amyloidosis (Robert Z. Orlowski, Cancer Cell. 2013, 24(3)). According to a survey, nearly 86,000 patients will be diagnosed with myeloma each year, and about 63,000 patients will die from disease-related complications each year (Becker, 2011). Due to the aging of the population, it is expected that the number of myeloma cases will increase year by year. Like many cancers, multiple myeloma has no known cause and cannot be cured. Some treatments for multiple myeloma are similar to those for other cancers, such as chemotherapy or radiation therapy, stem cell transplantation or bone marrow transplantation, targeted therapy or biological therapy (George, 2014). Antibody-based cellular immunotherapy has been shown to have significant clinical benefits for patients with hematologic malignancies, particularly in B-cell Non-Hodgkin’s lymphoma. Although current therapies for multiple myeloma often cause remission, almost all patients eventually relapse. Effective immunotherapeutic agents for treating multiple myeloma are needed.

[0008] Chimeric antigen receptor T (CAR-T) cell therapy is an emerging and effective cancer immunotherapy, especially in hematologic malignancies. However, the application of CAR-T cells is hindered by side effects such as cytokine release syndrome and on-target, off-tumor toxicity (Yu et al., Molecular Cancer 18(1): 125 (2019)). Improved binding molecules and engineered cells are needed. For example, there is a need to develop stable and therapeutically effective BCMA binding molecules for more effective or efficient CAR-T therapy. 3. SUMMARY

[0009] In one aspect, provided herein is a 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, wherein the first BCMA binding moiety is a first anti-BCMA single domain antibody, and the second BCMA binding moiety is a second anti-BCMA sdAb; and wherein each of the first sdAb and the second sdAb is a VHH domain; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein (i) the first 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: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (ii) the second anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 72; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0010] In some embodiments, the first anti-BCMA sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9, and the second anti-BCMA sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0011] In some embodiments, the first anti-BCMA sdAb is N-terminal to the second anti-BCMA sdAb. In other embodiments, the first anti-BCMA sdAb is C-terminal to the second anti-BCMA sdAb.

[0012] In some embodiments, the transmembrane domain is from a molecule selected from the group consisting of CD8a, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0013] In some embodiments, the transmembrane domain is from CD8a or CD28.

[0014] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is from CD3 zeta.

[0015] In some embodiments, the intracellular signaling domain comprises a chimeric signaling domain (“CMSD”), wherein the CMSD comprises a plurality of immunoreceptor tyrosine-based activation motifs (“CMSD ITAMs”) optionally connected by one or more linkers (“CMSD linkers”). In some embodiments, the CMSD comprises, from N-terminus to C-terminus: an optional N-terminal sequence - CD3 delta ITAM - an optional first CMSD linker - CD3 epsilon ITAM - an optional second CMSD linker - CD3 gamma ITAM - an optional third linker - DAP12 ITAM - an optional C-terminal sequence. In some embodiments, the CMSD comprises the amino acid sequence of SEQ ID NO: 53.

[0016] In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain comprises a cytoplasmic domain of CD28 and / or a cytoplasmic domain of CD137.

[0017] In some embodiments, the CAR provided herein further comprises a hinge domain 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 from CD8a.

[0018] In some embodiments, the CAR provided herein further comprises a signal peptide at the N-terminus of the polypeptide. In some embodiments, the signal peptide is from CD8a.

[0019] In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-34.

[0020] In yet another aspect, provided herein is an isolated nucleic acid comprising a nucleic acid sequence encoding the CAR provided herein. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35-46.

[0021] In yet another aspect, provided herein is a vector comprising the isolated nucleic acid encoding the nucleic acid sequence encoding the CAR provided herein.

[0022] In yet another aspect, provided herein is an engineered immune effector cell comprising a CAR, an isolated nucleic acid, or a vector provided herein. In some embodiments, the immune effector cell is a T cell.

[0023] In some embodiments, the engineered immune effector cell provided herein further comprises an exogenous Nef protein. In some embodiments, the exogenous Nef protein is selected from the group consisting of SIV Nef, HIV1 Nef, HIV2 Nef, and subtypes thereof. In some embodiments, the exogenous Nef protein is a wild-type Nef. In other embodiments, the exogenous Nef protein is a mutant Nef. In some embodiments, the mutant Nef comprises one or more mutations in myristoylation site, N-terminal a-helix, tyrosine-based AP recruitment, CD4 binding site, acidic cluster, proline-based repeat sequence, PAK binding domain, COP I recruitment domain, di-leucine-based AP recruitment domain, V-ATPase and Raf-1 binding domain, or any combination thereof. In some embodiments, the mutant Nef is a mutant SIV Nef comprising the amino acid sequence of SEQ ID NO: 51 (mutant SIV Nef M116).

[0024] In yet another aspect, provided herein is a pharmaceutical composition comprising an engineered immune effector cell provided herein and a pharmaceutically acceptable carrier.

[0025] In yet another aspect, provided herein is a method of treating a disease or a disorder in a subject, comprising administering to the subject an effective amount of an engineered immune effector cell or a pharmaceutical composition provided herein.

[0026] In some embodiments, the disease or disorder is a cancer. In some embodiments, the disease or disorder is multiple myeloma (MM).

[0027] In yet another aspect, provided herein is an anti-BCMA single domain antibody (sdAb) comprising (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; or (ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 72; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0028] In some embodiments, the sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In other embodiments, the anti-BCMA sdAb comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0029] In some embodiments, the anti-BCMA sdAb is a camelid sdAb. In other embodiments, the anti-BCMA sdAb is a humanized sdAb.

[0030] In yet another aspect, provided herein is an isolated nucleic acid or vector comprising a nucleic acid encoding an anti-BCMA sdAb provided herein.

[0031] In yet another aspect, provided herein is a chimeric antigen receptor (CAR) comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising an anti-BCMA sdAb provided herein; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0032] In yet another aspect, provided herein is an isolated nucleic acid or vector comprising a nucleic acid sequence encoding a CAR provided herein.

[0033] In yet another aspect, provided herein is an engineered immune effector cell comprising a CAR, an isolated nucleic acid or vector provided herein. In some embodiments, the immune effector cell is a T cell.

[0034] In yet another aspect, provided herein is a pharmaceutical composition comprising an engineered immune effector cell provided herein and a pharmaceutically acceptable carrier.

[0035] In yet another aspect, provided herein is a method of treating a disease or disorder in a subject comprising administering to the subject an effective amount of an engineered immune effector cell or a pharmaceutical composition provided herein. 4. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1Specific cytotoxicity of BCMA CAR-T cells (GSI5021 and LIC948A22 CAR-T cells) against human multiple myeloma cell line RPMI8226.Luc at different E:T ratios of 5:1 and 1 :1, respectively, is shown. "UnT" indicates untransduced T cells used as a control.

[0037] Figure 2 In vivo efficacy of GSI5021 and LIC948A22 CAR-T cells is shown. NCG mice were engrafted with human multiple myeloma cell line RPMI8226.Luc and treated 14 days later with HBSS, untransduced T cells (UnT), LIC948A22 CAR-T cells and GSI5021 CAR-T cells, respectively (indicated as day 0). Mice were evaluated by bioluminescence imaging at day -1 and weekly from day 0 to monitor tumor growth.

[0038] Figure 3 Specific cytotoxicity of humanized (LIC948A22H31 - LIC948A22H37) and non-humanized BCMA CAR-T cells (LIC948A22) against human multiple myeloma cell line RPMI8226.Luc at different E:T ratios of 2:1, 1 :1 and 1 :2, respectively, is shown. "UnT" indicates untransduced T cells used as a control.

[0039] Figure 4 In vivo efficacy of humanized (LIC948A22H34 and LIC948A22H37) and non-humanized BCMA CAR-T cells (LIC948A22) is shown. NCG mice were engrafted with human multiple myeloma cell line RPMI8226.Luc and treated 14 days later with HBSS, untransduced T cells (UnT), LIC948A22H34 CAR-T cells, LIC948A22H37 CAR-T cells and LIC948A22 CAR-T cells, respectively (indicated as day 0). Mice were evaluated by bioluminescence imaging at day -1 and weekly from day 0 to monitor tumor growth.

[0040] Figure 5 TCRaP expression of T cells transduced with lentivirus encoding LUC948A22 UCAR, LUC948A22H34, LUC948A22H36 and LUC948A22H37, respectively, is shown. "UnT" indicates untransduced T cells used as a control.

[0041] Figure 6The relative killing efficiency of T cells expressing LUC948A22 UCAR, LUC948A22H34, LUC948A22H36 and LUC948A22H37, respectively, against the multiple myeloma cell line RPMI8226.Luc at different E:T ratios of 5:1, 2.5:1 and 1.25:1 is shown. "UnT" indicates untransduced T cells and serves as a control. 5. DETAILED DESCRIPTION

[0042] The present disclosure is based, in part, on novel single-domain antibodies and chimeric antigen receptors that bind to BCMA or engineered cells comprising the same and their improved properties.

[0043] 5.1 DEFINITIONS

[0044] The techniques and procedures described or referenced herein include those commonly used and / or well understood techniques and procedures in the art such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Abitar ed. 2010); and Antibody Engineering Volumes 1 and 2 (Kontermann and Du¨bel eds., 2nd ed. 2010). Unless otherwise defined, the technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. To the extent that there is a conflict between the description set forth herein and that of any document incorporated herein by reference, the description set forth herein will control. In describing the present disclosure, the following terminology will be used and is intended to have the meanings attributed to them:

[0045] The terms "antibody," "immunoglobulin" or "Ig" are used interchangeably herein and are used in the broadest sense and specifically encompass, e.g., monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monopeptopic specificity formed from at least two intact antibodies, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single chain antibodies, and fragments thereof (e.g., domain antibodies), as described below. Antibodies can be human, humanized, chimeric and / or affinity matured, as well as antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include a polypeptide product of a B-cell that is capable of binding to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Fundamental Immunology (Paul, W., ed., 3rded. 1993) and Antibody Engineering (Borrebaeck, ed., 2nd ed. 1995); and Kuby, Immunology (3rd ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including those from camelid species (e.g., llama or alpaca) or humanized variants thereof, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, by which is meant a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments (e.g., antigen binding fragments) include single chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules containing an antigen binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found in, e.g., Harlow and Lane, Antibodies:A Laboratory Manual (1989); Mol.Biology and Biotechnology:A Comprehensive Desk Reference (Myers, ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule. An antibody can be an agonist antibody or an antagonist antibody. An antibody can be neither agonist nor antagonist.

[0046] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, a target antigen is a polypeptide. In certain embodiments, an antigen is associated with a cell, e.g., present on or in a cell.

[0047] An "intact" antibody is one that comprises an antigen binding site as well as a CL and at least heavy chain constant regions CHI, CH2 and CH3. The constant regions can include human constant regions or amino acid sequence variants thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0048] A "single-chain Fv" also abbreviated "sFv" or "scFv" is an antibody fragment that comprises the VH and VL antibody domains connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv fragments see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0049] The term "heavy chain only antibody" or "HCAb" refers to a functional antibody that comprises a heavy chain but lacks the light chain normally found in a 4-chain antibody. HCAbs are known to be produced by camelids (e.g., camels, llamas, or alpacas).

[0050] As used herein, a "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain and is capable of binding to an antigen (e.g., a single domain antibody that binds to BCMA). Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies that naturally lack light chains, such as antibodies from camelid species (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. For example, single domain antibodies can be derived from antibodies produced in camelid species, such as in camels, llamas, alpacas, vicunas, and guanacos. Other species outside of camelids can produce heavy chain antibodies that naturally lack light chains; VHHs derived from such other species are within the scope of the present disclosure. In some embodiments, the single domain antibodies (e.g., VHHs) provided herein have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., agent) as described herein. Single domain antibodies can be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0051] The term "binds" or "binding" refers to the interaction between molecules, including, for example, the formation of a complex. The interaction can be, for example, a noncovalent interaction including a hydrogen bond, an ionic bond, a hydrophobic interaction, and / or a van der Waals interaction. A complex can also include the association of two or more molecules held together by covalent or noncovalent bonds, interactions, or forces. The strength of the total noncovalent interaction between a single antigen binding site on an antibody and a single epitope of a target molecule (e.g., antigen) is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k off ) of a binding molecule (e.g., an antibody) from a monovalent antigen is the ratio of the rate constants for the dissociation (k on ) and association (k off ) of the molecule with the antigen (k on / k D ). The dissociation constant K D , which is inversely related to affinity, is the ratio of the rate constants for dissociation (k D ) and association (k on ). The lower the K off value, the higher the affinity of the antibody. K DThe affinity can be determined using any of the methods provided herein or any other methods well known to those skilled in the art. The affinity of a binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants (e.g., a multivalent antigen) comes into contact with an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site increases the likelihood of a reaction occurring at a second site. The strength of this multiple interaction between a multivalent antibody and an antigen is called affinity.

[0052] Regarding the binding molecules described herein, terms such as "bound to," "specifically bound to," and similar terms may be used interchangeably throughout this document. They refer to binding molecules, such as polypeptides, that specifically bind to the antigen-binding domain of an antigen. Binding molecules or antigen-binding domains that bind to or specifically bind to antigens can be identified, for example, by immunoassay. , Or other techniques known to those skilled in the art may be used for identification. In some embodiments, such as those measured using experimental techniques, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), the binding molecule or antigen-binding domain binds to or specifically binds to the antigen when the affinity of the binding molecule or antigen-binding domain for the antigen is higher than that for any cross-reactive antigen. Typically, the specific or selective reaction will be at least twice the background signal or noise, and may be more than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd edition, 1989). In some embodiments, as determined, for example by fluorescence-activated cell sorting (FACS) analysis or RIA, the binding degree of the binding molecule or antigen-binding domain to a "non-target" protein is less than about 10% of the binding degree of the binding molecule or antigen-binding domain to its specific target antigen. Binding molecules or antigen-binding domains that bind to antigens include those capable of binding antigens with sufficient affinity such that the binding molecule can be used as, for example, a therapeutic agent and / or diagnostic agent targeting the antigen. In some embodiments, the binding molecule or antigen-binding domain bound to the antigen has a dissociation constant (KB) less than or equal to 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. D In some embodiments, the binding molecule or antigen-binding domain binds to an antigenic epitope that is conserved in antigens from different species.

[0053] In certain embodiments, a binding molecule or antigen binding domain can comprise a "chimeric" sequence in which a portion of the heavy and / or light chain is identical with, or homologous to, a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with, or homologous to, a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81 :6851-55). Chimeric sequences can include humanized sequences.

[0054] In certain embodiments, a binding molecule or antigen binding domain can comprise a "humanized" form of a non-human (e.g., camelid, murine, non-human primate) antibody, which includes sequences from a human immunoglobulin (e.g., an acceptor antibody) in which native CDR residues are replaced by corresponding CDR residues from a non-human species (e.g., a donor antibody), e.g., a camelid, mouse, rat, rabbit, or non-human primate having a desired specificity, affinity, and capability. In some cases, one or more FR region residues of the human immunoglobulin sequences are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the antibody performance. A humanized antibody heavy or light chain can comprise substantially the entire at least one or more variable regions, wherein all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos.: 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0055] In some embodiments, the binding molecule or antigen-binding domain may comprise a portion of a “fully human antibody” or “human antibody,” wherein these terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. The binding molecule may comprise a single-domain antibody sequence. In specific embodiments, these terms refer to an antibody comprising both a human-derived variable region and a constant region. In some embodiments, a “fully human” antibody may also encompass an antibody that binds a polypeptide and is encoded by a nucleic acid sequence of a naturally occurring somatic variant of a human immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human immunoglobulin sequences, as in Kabat et al. (see Kabat et al. (1991)). Sequences of Proteins of Immunological Interest As described in the fifth edition of the National Health and Human Services Journal (NIH Publication No. 91-3242), a “human antibody” is an antibody having an amino acid sequence corresponding to that of human-produced antibodies and / or having been manufactured using any technology for manufacturing human antibodies. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68 (2006)). The methods described below can also be used to prepare human monoclonal antibodies: Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, for example, mice (regarding XENOMOUSE). TMTechniques, see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Patent Nos. 6,075,181 and 6,150,584). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006) for human antibodies generated via human B-cell hybridoma technology.

[0056] In certain embodiments, a binding molecule or antigen binding domain can comprise portions of a "recombinant human antibody," where the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic for and / or transchromosomal for human immunoglobulin genes (see e.g., Taylor, L. D. et al., Nucl. Acids Res. 20:6287-6295 (1992)) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, such recombinant human antibodies can be mutagenized in vitro (or, when using animals transgenic for human Ig sequences, mutagenized in vivo in somatic cells), and thus the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, can not naturally occur within the human antibody germline library in vivo. Sequences of Proteins of Immunological Interest

[0057] ​In certain embodiments, a binding molecule or antigen binding domain can comprise a portion of a "monoclonal antibody," where the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts or for known post- translational modifications such as isomerization or deamidation of amino acids, oxidation of methionine, or deamidation of asparagine or glutamine, each of which will recognize a single epitope on an antigen. In particular embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for making the antibody. For example, the monoclonal antibodies useful in the present disclosure can be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Patent 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991), for example. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are also well known in the art. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) at pp.59-103. Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0058] A typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus a variable region (VH), followed by three constant regions (CH) each. Each L chain has a variable region at its N-terminus (VL), followed by a constant region (CL) at its other end. The VL is aligned with the VHand the CL is aligned with the first constant region of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing between a VHand a VLtogether forms the single antigen-binding site of a Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5th ed. 2001).

[0059] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. A conventional IgG typically comprises two Fab regions, one on each of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable and one constant region of each of a heavy and light chain. More specifically, the variable and constant regions of the heavy chain in a Fab region are the VH and CHI regions, and the variable and constant regions of the light chain in a Fab region are the VL and CL regions. The VH, CHI, VL, and CL in a Fab region can be arranged in various ways to confer antigen binding ability according to the present disclosure. For example, the VH and CHI regions can be on one polypeptide, and the VL and CL regions can be on a separate polypeptide, similar to the Fab regions of a conventional IgG. Alternatively, the VH, CHI, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the subsections below.

[0060] The term "variable region," "variable domain," "V region," or "V domain" refers to the portion of a light or heavy chain of an antibody that is typically located at the amino-terminus of the light or heavy chain and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is involved in binding and specificity of each particular antibody to its particular antigen. The variable region of the heavy chain can be referred to as "VH." The variable region of the light chain can be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies, and are used to bind to, and specifically for, an antigen. However, the variability is not evenly distributed throughout the 110-amino acid span of the variable region. Instead, it is concentrated in three segments called "hypervariable regions" both in the light chain and the heavy chain that are referred to as "complementarity-determining regions" (CDRs). The more highly conserved flanking stretches of the variable region are referred to as "framework regions" (FRs). From N- to C-terminus, both light and heavy chains have four FR regions, generally termed FR1, FR2, FR3, and FR4. Within each variable region, the CDRs are separated by the FR regions, which contribute to the overall variable region structure. The CDRs are primarily involved in antigen recognition. The V region of the light chain and the V region of the heavy chain are aligned at the FR1 region of each chain, and contribute to the formation of the antigen binding site. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). The V region of the light chain and the V region of the heavy chain are aligned at the FR1 region of each chain, and contribute to the formation of the antigen binding site. Sequences of Proteins of Immunological Interest (5th Ed. 1991)). The constant regions are not directly involved in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The sequence of the variable region differs greatly among different antibodies. In particular embodiments, the variable region is a human variable region.

[0061] The terms "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system for the heavy chain variable region or light chain variable region used in the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domains. For example, a heavy chain variable domain can include a single amino acid insertion after residue 52 (according to Kabat, residue 52a) and three insertions of three amino acids each after residue 82 (e.g., residues 82a, 82b, and 82c, according to Kabat, etc.). The Kabat residue numbering of a given antibody can be determined by alignment of the consensus regions of the antibody sequence with the "standard" Kabat numbered sequence. The Kabat numbering system is typically used when referring to residues in the variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is typically used when referring to residues in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, AbM, Chothia, Contact, IMGT, and AHon.

[0062] The term "heavy chain" when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and the carboxy-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be of one of five different classes, e.g., isotypes, called alpha (a), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different classes of heavy chains vary in size: a, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. These different classes of heavy chains give rise to five well-known classes of antibodies (e.g., isotypes), namely IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4, when combined with light chains.

[0063] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different classes, called kappa (κ) or lambda (λ).

[0064] As used herein, the terms "hypervariable region," "HVR," "complementarity determining region," and "CDR" are used interchangeably. "CDR" refers to one of three hypervariable regions (H1, H2, or H3) within the non- framework region of an immunoglobulin (Ig or antibody) VH beta-sheet framework, or one of three hypervariable regions (L1, L2, or L3) within the non- framework region of an antibody VL beta-sheet framework. Thus, CDRs are sequences that are interspersed between framework sequences.

[0065] CDR regions are well known to those skilled in the art and have been defined by well- known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra; Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia instead refers to the location of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Volume 2 (Kontermann and Dubel eds., 2nded. 2010). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and is widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003)). IMGT is an integrated information system for the study of immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complex (MHC) in human and other vertebrates. In this text, CDRs are referred to according to the amino acid sequence and position in the light or heavy chain. Since the "positions" of CDRs within immunoglobulin variable domain structures are conserved between species and occur in structures called loops, it is easy to identify CDR and framework residues by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from one species of immunoglobulin into a recipient framework, often from a human antibody. A further numbering system (AHon) was developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-70 (2001). Correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, are well known to those of skill in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are illustrated in Table 1 below.

[0066] Table 1. Exemplary CDRs according to various numbering systems

[0067]

[0068]

[0069] The boundaries of a given CDR can vary depending on the scheme used to identify it. Thus, unless otherwise specified, the terms "CDR" and "complementarity determining region" with reference to a given antibody or region thereof, e.g., a variable region, as well as individual CDRs of an antibody or region thereof (e.g., CDR-H1, CDR-H2), are understood to encompass complementarity determining regions as defined by any of the known schemes described above. In some cases, the scheme used to identify a particular CDR or CDRs is specified, e.g., CDRs defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, a particular amino acid sequence of a CDR is given. It should be noted that CDR regions can also be defined by a combination of various numbering systems, e.g., a combination of the Kabat and Chothia numbering systems or a combination of the Kabat and IMGT numbering systems. Thus, for example, the term "CDR as set forth in a particular VH or VHH" includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereto. Once a variable region (e.g., VHH, VH, or VL) is given, one of skill in the art will understand that the CDRs within that region can be defined by different numbering systems or combinations thereof.

[0070] Hypervariable regions can include the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.

[0071] The term "constant region" or "constant domain" refers to the carboxy-terminal portion of the light and heavy chains that is not directly involved in binding of an antibody to an antigen but exhibits various effector functions, such as interaction with the Fc receptor. The term refers to the portion of an immunoglobulin molecule that, in contrast to the variable region that contains the antigen binding site, has a more conserved amino acid sequence. The constant region can contain the CH1, CH2 and CH3 regions of the heavy chain and the CL region of the light chain.

[0072] The term "framework" or "FR" refers to those variable region residues flanking the CDRs. FR residues are found, for example, in chimeric antibodies, humanized antibodies, human antibodies, domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0073] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or Pro230, to the carboxy-terminus. The C-terminal lysine (residue 447 according to EU numbering system) of the Fc region can be removed, e.g., during production or purification of the antibody, or by engineering the nucleic acid encoding the antibody heavy chain recombinantly. Therefore, the composition of complete antibodies can include a population of antibodies with all K447 residues removed, a population of antibodies with no K447 residues removed, and a mixture of antibodies with and without K447 residues. A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of B cell receptor; and the like. Such effector functions typically require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain), and can be assessed using various assays known to the skilled artisan. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of having at least one amino acid modification, e.g., substitution, addition, or deletion. In certain embodiments, a variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., between about 1 and about 10 amino acid substitutions, or between about 1 and about 5 amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide. A variant Fc region herein can have at least about 80% homology to a native sequence Fc region and / or to the Fc region of a parent polypeptide, or at least about 90% homology thereto, e.g., at least about 95% homology thereto.

[0074] As used herein, an "epitope" is a term of art in the field and refers to a local region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. For example, in the case of a polypeptide antigen, an epitope can be a contiguous amino acid of the polypeptide ("linear" epitope), or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide ("conformational," "non-linear," or "discontinuous" epitope). Those skilled in the art will appreciate that, in general, a linear epitope can or can not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a set of amino acids regardless of whether they are folded into a native three-dimensional protein structure. In other embodiments, a binding molecule requires that the amino acid residues making up an epitope exhibit a particular conformation (e.g., bend, twist, flip, or fold) in order to recognize and bind that epitope.

[0075] A "blocking" antibody or an "antagonist" antibody is one which inhibits or decreases the biological activity of the antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

[0076] An "agonist" or activating antibody is one which enhances or initiates signaling by the antigen to which it binds. In some embodiments, an agonist antibody causes or activates signaling in the absence of the natural ligand.

[0077] "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent 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 various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve the maximum alignment over the full length of the sequences being compared.

[0078] The term "specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen binding protein (e.g., CAR or sdAb). For example, a native antibody is monospecific. As used herein, the term "multispecific" indicates that an antigen binding protein (e.g., CAR or sdAb) has two or more antigen binding sites, at least two of which bind different antigens. As used herein, the term "bispecific" indicates that an antigen binding protein (e.g., CAR or sdAb) has two different antigen binding specificities. As used herein, the term "monospecific" CAR indicates an antigen binding protein (e.g., CAR or sdAb) having one or more binding sites, each of which binds the same epitope of an antigen.

[0079] As used herein, the term "valency" indicates the presence of a specified number of binding sites in an antigen binding protein (e.g., CAR or sdAb). For example, a native antibody or full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate the presence of two binding sites, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding protein (e.g., CAR or sdAb).

[0080] As used herein, a "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to graft one or more antigen specificities onto an immune effector cell such as a T cell. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immunoreceptors." In some embodiments, a CAR comprises an extracellular antigen binding domain with specificity for one or more antigens (e.g., tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. A "CAR-T cell" refers to a T cell that expresses a CAR.

[0081] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms embrace not only the purely polypeptide, but also, for example, polypeptides with one or more non-amino acid(s) added to the polymer, for example, PEG. Polypeptides can be modified by natural processes, such as post-translational modifications, or by intervention resulting in the addition of a moiety to the polypeptide. It is understood that a polypeptide of the present disclosure can be based on an antibody or other member of the immunoglobulin superfamily, and thus in certain embodiments, a "polypeptide" can occur as a single chain or as two or more associated chains.

[0082] "Polynucleotide" or "nucleic acid" as used interchangeably herein refer to polymers of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. An "oligonucleotide" as used herein refers to a short, usually single-stranded synthetic polynucleotide, which is usually, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of a polynucleotide applies equally to an oligonucleotide. Cells that produce binding molecules of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Unless otherwise indicated, the left-hand end of any single-stranded polynucleotide sequence is the 5' terminus; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcriptional direction; sequence regions on the DNA strand having the same sequence as the RNA transcript are referred to as "upstream sequences" when located 5' to the 5' terminus of the RNA transcript; "downstream sequences" when located 3' to the 3' terminus of the RNA transcript.

[0083] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, with which the sequence naturally occurs. An "isolated" nucleic acid molecule is one that is separated from the other nucleic acid molecules of the cell of the natural source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a particular embodiment, one or more nucleic acid molecules encoding a single domain antibody or antibody as described herein are isolated or purified. The term includes nucleic acid sequences that have been removed from their naturally occurring environment and includes recombinant or cloned DNA molecules and chemical syntheses or biosynthetic analogs produced by heterologous systems. A substantially pure molecule can include an isolated form of the molecule. In particular, an "isolated" nucleic acid molecule encoding a CAR or sdAb described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which the isolated nucleic acid molecule is produced.

[0084] Unless otherwise specified, "nucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences, which play degenerate versions of each other and encode the same amino acid sequence. To the extent that a nucleotide sequence encoding a protein can contain one or more introns in certain versions, the phrase nucleotide sequence encoding a protein or RNA can also include introns.

[0085] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are appropriate for a given host organism are readily identifiable and are well known in the art. For example, appropriate control sequences for prokaryotic include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0086] As used herein, the term "operably linked" and similar phrases (e.g., in genetic fusion) when used in reference to nucleic acids or amino acids refer to the functional linkage of the nucleic acid sequences or amino acid sequences to each other such that they perform a function together. For example, operably linked promoter, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, operably linked peptides are peptides in which functional domains are placed at appropriate distances from each other to impart the intended function of each domain.

[0087] The term "vector" refers to an agent used to carry or comprise a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, to introduce the nucleic acid sequence into a host cell. Useful vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include a selectable sequence or marker useful for stable integration into the host cell chromosome. In addition, the vector can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included provide, for example, resistance to an antibiotic or toxin, complement a nutritional deficiency, or provide a critical nutrient not present in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., antibody heavy and light chains or antibody VH and VL), the two nucleic acid molecules can be inserted, for example, into a single expression vector or separate expression vectors. For single vector expression, the encoding nucleic acids are operably linked to a common expression control sequence or to different expression control sequences, e.g., an inducible promoter and a constitutive promoter. Confirmation of the introduction of the nucleic acid molecule into the host cell can be made using methods well known in the art. Such methods include, for example, nucleic acid analysis, e.g., Northern blot or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable assays to assess expression of the introduced nucleic acid sequence or its corresponding gene product. One of skill in the art understands that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product, and further understands that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0088] The term "host" as used herein refers to an animal, such as a mammal (e.g., a human).

[0089] The term "host cell" as used herein refers to a particular subject cell that can be transfected with a nucleic acid molecule, as well as the progeny or potential progeny of such a cell. Progeny can not be identical to the parent cell from which they were derived, due to mutations that occur during replication or mutations and environmental influences that are introduced during propagation in a host cell.

[0090] The term "autologous" as used herein means any material derived from the same individual to which it is subsequently reintroduced.

[0091] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0092] As used herein, the term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into a 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 subject cell and its progeny.

[0093] The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. United States Pharmacopeia 、 European Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0094] "Excipient" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material. Excipients include, for example, encapsulating material or additives, such as absorption accelerators, antioxidants, binders, buffering agents, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfuming agents, preservatives, propellants, release agents, sterilants, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freunds' adjuvant (complete or incomplete) or vehicle.

[0095] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG), and PLURONICS TM . Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, The Science and Practice of Pharmacy, (18th Edition 1990). Remington's Pharmaceutical Sciences (18th Edition 1990).

[0096] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenecity, or other problems or complications commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6thEdition; Rowe et al. eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rdEdition; Ash and Ash eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2ndEdition; Gibson ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is non-toxic to the cells or mammals exposed thereto at the doses and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0097] In some embodiments, the excipient is a sterile liquid such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. The excipient can further include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral composition, including formulation, can include standard excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0098] The composition, including the pharmaceutical compound, can contain, for example, the binding molecule (e.g., antibody) in isolated or purified form, in an amount, together with the excipient(s).

[0099] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic molecule of a single-domain antibody or a combination agent and single-domain antibody or pharmaceutical composition provided herein that is sufficient to effect a desired result.

[0100] The terms "subject" and "patient" are used interchangeably herein. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., a human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, such as a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, such as a human, at risk of developing a disease or disorder.

[0101] "Administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance that is present outside of the body into the body of a patient, for example, by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0102] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or disorder resulting from the administration of one or more therapies. Treatment can be determined by assessing whether one or more symptoms associated with an underlying disorder are reduced, alleviated, and / or ameliorated, such that an improvement is observed in a patient, although the patient can still suffer from the underlying disorder. The term "treatment" includes both control and improvement of a disease. The terms "manage," "managing," and "management" refer to the beneficial effects obtained with a therapy in a subject, which do not necessarily cure the disease.

[0103] The terms "prevent," "preventing," and "prevention" refer to the reduction in the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (e.g., diabetes or cancer).

[0104] As used herein, "delaying development of cancer" means postponing, hindering, slowing, retarding, stabilizing, and / or postponing the development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As will be apparent, a sufficient or significant delay can in effect result in a prevention, in that the individual does not, in fact, develop the disease. A method that "delays development of cancer" is one that reduces the likelihood of development of the disease within a given timeframe and / or reduces the extent of the disease within a given timeframe as compared to not using the method. Such comparisons are typically based on clinical studies using statistically significant numbers of individuals. Development of cancer can be detected using standard methods, including, but not limited to, computed axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation testing, arteriography, or biopsy. Development can also refer to progression of a cancer that can not initially be detectable, including occurrence, recurrence, and onset.

[0105] As used herein, "B cell-related disease or disorder" means a disease or disorder mediated by B cells or conferred by aberrant B cell function, e.g., dysregulation of B cell function. As used herein, "B cell-related disease or disorder" includes, but is not limited to, B cell malignancies, e.g., B cell leukemia or B cell lymphoma. It also includes marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, primary mediastinal B cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia (B-PLL), follicular lymphoma (FL), burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), precursor B-lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), high grade B cell lymphoma (HGBL), and multiple myeloma (MM). "B cell-related disease or disorder" also includes certain autoimmune and / or inflammatory diseases, e.g., those associated with inappropriate or enhanced B cell numbers and / or activation.

[0106] As used herein, "BCMA-related disease or disorder" means a disease or disorder comprising cells or tissues in which BCMA is expressed or overexpressed. In some embodiments, a BCMA-related disease or disorder comprises cells in which BCMA is aberrantly expressed. In other embodiments, a BCMA-related disease or disorder comprises cells that lack BCMA internally or on their surface.

[0107] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0108] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0109] It should be understood that whenever a numeral is used in this document, it is intended to represent a broad category of values, for example, a range of values. For example, a value of "10" is intended to represent a broad category of values including 10, 11, 12, 13, 14, 15, etc. Whenever the term "comprising" is used in this document, it is intended to provide a broad category of embodiments including other similar embodiments described in terms of "consisting of" and / or "consisting essentially of." It should be understood that whenever the term "consisting essentially of is used in this document, it is intended to provide a broad category of embodiments including other similar embodiments described in terms of "consisting of."

[0110] The term "between" as used in a phrase such as "between A and B" or "between A-B" means a range that includes both A and B.

[0111] The term "and / or" as used in a phrase such as "A and / or B" as used herein is intended to include both A and B, A or B, A (alone), and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0112] 5.2. Single Domain Antibodies

[0113] 5.2.1. Single Domain Antibodies that Bind to BCMA

[0114] In one aspect, provided herein are single domain antibodies (e.g., humanized VHH domains) that are capable of binding to BCMA.

[0115] In some embodiments, the single domain antibodies (e.g., VHH domains) provided herein bind to human BCMA. In some embodiments, the anti-BCMA single domain antibodies provided herein modulate one or more BCMA activities. In some embodiments, the anti-BCMA single domain antibodies provided herein are antagonist antibodies.

[0116] In some embodiments, the anti-BCMA single domain antibodies provided herein bind to BCMA (e.g., human BCMA) with a dissociation constant (K D ) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10 -8M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure, including by RIA, e.g., with Fab versions of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by bio-layer interferometry (BLI) or surface plasmon resonance (SPR) assays, by using, e.g., Octet® Red96 system, or by using, e.g., Octet® TM-2000 or TM-3000. The "on-rate" or "rate of association" or "association rate" or "kon" can also be measured using the same bio-layer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, using, e.g., Octet® Red96, TM-2000 or TM-3000 systems.

[0117] In some embodiments, the anti-BCMA single domain antibodies provided herein are VHH domains. Exemplary VHH domains provided herein were generated as described in Section 6, below, including VHH domains designated 269A37948H3, 269AS34822H1, 269AS34822H2, 269AS34822H3, 269AS34822H4, 269AS34822H5, 269AS34822H6, 269AS34822H7, as also shown in Table 4, below.

[0118] Accordingly, in some embodiments, the single domain antibodies provided herein comprise one or more CDR sequences of any of 269A37948H3, 269AS34822H1, 269AS34822H2, 269AS34822H3, 269AS34822H4, 269AS34822H5, 269AS34822H6, 269AS34822H7. In some embodiments, provided herein are single domain antibodies that bind to BCMA comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected from those sequences in 269A37948H3, 269AS34822H1, 269AS34822H2, 269AS34822H3, 269AS34822H4, 269AS34822H5, 269AS34822H6, 269AS34822H7. The CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the anti-BCMA single domain antibodies are of Camelidae. In some embodiments, the anti-BCMA single domain antibodies are humanized. In some embodiments, the anti-BCMA single domain antibodies comprise an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0119] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 1; CDR2 and comprises the amino acid sequence of SEQ ID NO: 2; and CDR3 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-BCMA single domain antibodies are of Camelidae. In some embodiments, the anti-BCMA single domain antibodies are humanized. In some embodiments, the anti-BCMA single domain antibodies comprise an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0120] In other embodiments, provided herein is a single domain antibody that binds to BCMA, the single domain antibody comprising: (i) a CDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1; (ii) a CDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, and (iii) a CDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the anti-BCMA single domain antibody is of Camelid origin. In some embodiments, the anti-BCMA single domain antibody is humanized. In some embodiments, the anti-BCMA single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0121] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 4; the CDR2 comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 72; and the CDR3 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-BCMA single domain antibody is of Camelid origin. In some embodiments, the anti-BCMA single domain antibody is humanized. In some embodiments, the anti-BCMA single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0122] In other embodiments, provided herein is a single domain antibody that binds to BCMA, the single domain antibody comprising: (i) a CDR1 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4; (ii) a CDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5, or a CDR2 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 72; and (iii) a CDR3 comprising an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the anti-BCMA single domain antibody is of Camelid origin. In some embodiments, the anti-BCMA single domain antibody is humanized. In some embodiments, the anti-BCMA single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0123] In some embodiments, the single domain antibody further comprises one or more framework regions of 269A37948H3, 269AS34822H1, 269AS34822H2, 269AS34822H3, 269AS34822H4, 269AS34822H5, 269AS34822H6, and / or 269AS34822H7. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 9. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 10. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 11. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 12. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 13. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 14. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 15. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 16.

[0124] In some embodiments, the single domain antibodies provided herein are humanized single domain antibodies. In some embodiments, the humanized single domain antibodies can be generated using the methods exemplified in Section 6 below or the methods described in the following section.

[0125] The framework regions described herein are determined according to the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, e.g., Kabat, IMGT, or Chothia, the framework regions are the amino acid residues in the variable region that surround the CDRs, in the following format from N- to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0126] In some embodiments, an isolated anti-BCMA single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, an isolated anti-BCMA single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 11. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, an isolated anti-BCMA single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 13. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, an isolated anti-BCMA single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 15. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 16.

[0127] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an amino acid sequence that is a certain percentage identical to any one of antibodies 269A37948H3, 269AS34822H1, 269AS34822H2, 269AS34822H3, 269AS34822H4, 269AS34822H5, 269AS34822H6, and 269AS34822H7.

[0128] A mathematical algorithm can be used to determine the percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). One non-limiting example of a mathematical algorithm that is used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set to default, e.g., score = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set to default, e.g., score = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search to detect distant relationships between molecules (Id.). When utilizing BLAST, gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm that is used to compare sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using similar techniques as described above, allowing for gaps or not. In calculating percent identity, typically only exact matches are counted.

[0129] In some embodiments, an anti-BCMA single domain antibody is provided, comprising a VHH domain having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16. In some embodiments, a VHH sequence having at least about 75%, 80%, 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 single domain antibody comprising that sequence retains the ability to bind to BCMA. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16. In some embodiments, the substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., in the FRs). Optionally, the anti-BCMA single domain antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16, including post-translational modifications of that sequence.

[0130] In certain embodiments, a single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9, wherein the single domain antibody binds to BCMA.

[0131] In certain embodiments, a single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, wherein the single domain antibody binds to BCMA.

[0132] In certain embodiments, a single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11, wherein the single domain antibody binds to BCMA. In certain embodiments, a single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11, wherein the single domain antibody binds to BCMA.

[0133] In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12, wherein the single domain antibody binds to BCMA.

[0134] In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, wherein the single domain antibody binds to BCMA.

[0135] In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14, wherein the single domain antibody binds to BCMA.

[0136] In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15, wherein the single domain antibody binds to BCMA.

[0137] In certain embodiments, the single domain antibody described herein comprises a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 16, wherein the single domain antibody binds to BCMA.

[0138] In some embodiments, epitopes can be mapped, e.g., by combinatorial alanine scanning, to identify amino acids in the BCMA protein that are necessary for interaction with the anti-BCMA single domain antibodies provided herein. In some embodiments, the conformation and crystal structure of an anti-BCMA single domain antibody that binds to BCMA can be employed to identify epitopes. In some embodiments, the present disclosure provides antibodies that bind to the same epitope as any of the anti-BCMA single domain antibodies provided herein. For example, in some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, an antibody is provided that binds to the same epitope as an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 16.

[0139] In some embodiments, provided herein is an anti-BCMA antibody or antigen-binding fragment thereof that specifically binds to BCMA in competition with any one of the anti-BCMA single domain antibodies described herein. In some embodiments, competitive binding can be determined using an ELISA assay. For example, in some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 15. In some embodiments, provided is an antibody that specifically binds to BCMA in competition with an anti-BCMA single domain antibody comprising the amino acid sequence of SEQ ID NO: 16.

[0140] In some embodiments, provided herein is a BCMA binding protein comprising any of the above-described anti-BCMA single domain antibodies. In some embodiments, the BCMA binding protein is a monoclonal antibody, including a camelid, chimeric, humanized, or human antibody. In some embodiments, the anti-BCMA antibody is an antibody fragment, such as a VHH fragment. In some embodiments, the anti-BCMA antibody is a heavy chain-only antibody comprising a full-length Fc region of any antibody class or isotype, such as IgGl or IgG4. In some embodiments, the Fc region has reduced or minimized effector function. In some embodiments, the BCMA binding protein is a fusion protein comprising an anti-BCMA single domain antibody provided herein. In other embodiments, the BCMA binding protein is a multi-specific antibody comprising an anti-BCMA single domain antibody provided herein. Other exemplary BCMA binding molecules are described in more detail in the following sections.

[0141] In some embodiments, an anti-BCMA antibody (e.g., anti-BCMA single domain antibody) or antigen binding protein according to any of the above embodiments can incorporate any of the features, alone or in combination, as described in Sections 5.2.2 to 5.2.7 below.

[0142] 5.2.2. Humanized single domain antibodies

[0143] The single domain antibodies described herein include humanized single domain antibodies. General strategies for humanizing single domain antibodies from camelid species have been described (see, e.g., Vincke et al., J. Biol. Chem., 284(5):3273-3284 (2009)) and can be used to generate humanized VHH domains as disclosed herein. Design of humanized single domain antibodies from camelid species can include signature residues in the VHH, e.g., residues 11, 37, 44, 45, and 47 (residue numbering according to Kabat) (Muyldermans, Reviews Mol Biotech 74:277-302 (2001).

[0144] Humanized antibodies, such as the humanized single domain antibodies disclosed herein, can also be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); and Roguska et al., PNAS 91 :969-973 (1994)), chain shuffling (U.S. Patent No. 5,565,332), and techniques such as those disclosed in U.S. Patent No. 6,407,213; U.S. Patent No. 5,766,886; WO9317105; Tan et al., J. Immunol. 169:1119 25 (2002); Caldas et al., Protein Eng. 13(5):353-60 (2000); Morea et al., Methods 20(3):267 79 (2000); Baca et al., J. Biol. Chem. 272(16): 10678-84 (1997); Roguska et al., Protein Eng. 9(10):895 904 (1996); Couto et al., Cancer Res. 55(23 Suppl):5973s-5977s (1995); Couto et al., Cancer Res. 55(8): 1717-22 (1995); Sandhu JS, Gene 150(2):409-10 (1994); and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Publication No. US 2005 / 0042664 Al (February 24, 2005), each of which is incorporated by reference in its entirety.

[0145] In some embodiments, the single domain antibodies provided herein can be humanized single domain antibodies that bind to BCMA, including human BCMA. For example, the humanized single chain antibodies of the present disclosure can comprise one or more CDRs set forth in SEQ ID NOs: 9-16. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can incorporate one or more amino acid residues from a non-human source. These non-human amino acid residues are typically referred to as "import" residues, which are typically taken from an "import" variable domain. For example, humanization can be performed by substituting hypervariable region sequences of the human antibody for the corresponding sequences of a non-human antibody, following the method herein or the methods taught by Jones et al., Nature 321 :522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Verhoeyen et al., Science 239:1534-36 (1988). In a particular embodiment, humanization of the single domain antibodies provided herein is performed as described in Section 6, infra.

[0146] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the CDRs of a parent non-human antibody are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in the CDRs actually contact antigen, and referred to these residues as "specificity determining residues" or SDRs (Padlan et al., FASEB J. 9:133-39 (1995)). In SDR grafting, only the SDR residues are grafted onto a human antibody framework (see, e.g., Kashmiri et al., Methods 36:25-34 (2005)).

[0147] The choice of human variable domain used to make the humanized antibody is important to reduce antigenicity. For example, according to a so-called "best-fit" method, the variable domain sequence of the non-human antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to the non-human antibody is chosen as the human framework for the humanized antibody (Sims et al., J. Immunol. 151 :2296-308 (1993); and Chothia et al., J. Mol. Biol. 196:901-17 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); and Presta et al., J. Immunol. 151 :2623-32 (1993)). In some cases, the framework is derived from a most-frequently occurring human subgroup V L6 Subgroup I (V L 6 I) and V H Subgroup III (V H III) consensus sequences. In another approach, human germline genes are used as a source of framework regions.

[0148] In an alternative paradigm for CDR comparison-based humanization, called superhumanization, FR homology is irrelevant. This approach consists of comparing the non-human sequence to the functional human germline gene repertoire. Genes that encode canonical structures identical or closely related to the murine sequence are then selected. Next, among the genes that share canonical structures with the non-human antibody, the genes with the highest homology within the CDRs are selected as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., J. Immunol. 169: 1119-25 (2002)).

[0149] It is also generally desirable to humanize antibodies while retaining affinity for the antigen and other favorable biological properties. To achieve this goal, according to one approach, humanized antibodies are made by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional modeling in conjunction with analysis of the parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These programs are for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-24 (2002)), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815 (1993)), and Swiss PDBViewer (Guex and Peitsch, Electrophoresis 18: 2714-23 (1997)). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and input sequences so that the desired antibody characteristics, such as increased affinity for the target antigen, are achieved. In general, the hypervariable region residues will directly and most substantially participate in influencing antigen binding.

[0150] Another approach to antibody humanization is based on an antibody humanness metric called Human String Content (HSC). This approach compares the mouse sequence to the human germline gene repertoire and scores the differences as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure, resulting in a variety of different humanized variants (Lazar et al., Mol. Immunol. 44: 1986-98 (2007)).

[0151] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods include those based on the generation of large humanized variant libraries and selection of the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23:1105-16 (2005); Dufner et al., Trends Biotechnol. 24:523-29 (2006); Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003); and Schlapschy et al., Protein Eng. Des. Sel. 17:847-60 (2004)).

[0152] In FR library methods, a series of residue variants are introduced at specific positions in the FRs, and the library is then screened to select the FRs that best support the grafted CDRs. The residues to be substituted can include some or all of the "thumbprint" residues identified as likely to contribute to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224:487-99 (1992)), or a more limited set of target residues identified from Baca et al. J. Biol. Chem. 272:10678-84 (1997).

[0153] In FR shuffling, the entire FR is combined with a non-human CDR rather than creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods 36:43-60 (2005)). A one-step FR shuffling method can be used. This method has been shown to be effective as the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60 (2007)).

[0154] The "humanization" approach is based on experimental identification of the minimal specificity determinants (MSDs) and based on sequential replacement of non-human fragments into a human FR library and evaluation of binding. This approach can typically preserve the epitope and identify antibodies from multiple subgroups of different human V segment CDRs.

[0155] "Human engineering" methods include altering a non-human antibody or antibody fragment by making specific changes to the amino acid sequence of the antibody to produce a modified antibody that has reduced immunogenicity in humans, but still retains the desired binding properties of the original non-human antibody. Typically, this technique involves classifying the amino acid residues of the non-human antibody as "low risk," "moderate risk," or "high risk" residues. Classification is performed using a global risk / reward calculation that assesses the predicted benefit (e.g., for immunogenicity in humans) of making a particular substitution against the risk that the substitution will affect the resulting antibody fold. A particular human amino acid residue to be substituted at a given position (e.g., low risk or moderate risk) in the non-human antibody sequence can be selected by aligning the amino acid sequence from the non-human antibody variable region with the corresponding region of a particular or consensus human antibody sequence. According to the alignment, an amino acid residue in the low risk or moderate risk position in the non-human sequence can be substituted for the corresponding residue in the human antibody sequence. Techniques for making human engineered proteins are described in more detail in: Studnicka et al., Protein Engineering 7:805-14 (1994); U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619; and PCT Publication WO 93 / 11794.

[0156] Composite Human Antibodies TM Composite Human Antibodies can be generated using, for example, the XenoMouse® technology (Antitope Ltd., Cambridge, United Kingdom). To generate Composite Human Antibodies, variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibodies.

[0157] Deimmunized antibodies are antibodies that have been stripped of T cell epitopes. Methods for making deimmunized antibodies have been described. See, e.g., Jones et al., Methods Mol Biol. 525:405-23 (2009); xiv and De Groot et al., Cell. Immunol. 244:148-153 (2006)). Deimmunized antibodies comprise T cell epitope-depleted variable regions and human constant regions. Briefly, the variable region of an antibody is cloned and then T cell epitopes are identified by testing overlapping peptides derived from the antibody variable region in a T cell proliferation assay. T cell epitopes are identified via computational methods to identify peptides that bind to human MHC class II. Mutations are introduced in the variable region to eliminate binding to human MHC class II. The mutated variable region is then utilized to produce a deimmunized antibody.

[0158] 5.2.3. Single Domain Antibody Variants

[0159] In some embodiments, amino acid sequence modifications of the single domain antibodies described herein that bind to BCMA are contemplated. For example, it can be desirable to optimize binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the single domain antibodies described herein that bind to BCMA, variants of the single domain antibodies described herein that bind to BCMA are contemplated. For example, single domain antibody variants can be prepared by introducing appropriate nucleotide changes into the coding DNA, and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art appreciate that amino acid changes can alter post-translational processes of the single domain antibody.

[0160] Chemical modification

[0161] In some embodiments, the single domain antibodies provided herein are chemically modified, e.g., by covalent attachment of any type of molecule to the single domain antibody. Antibody derivatives can include antibodies that are chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or conjugation with one or more immunoglobulin domains (e.g., an Fc or a portion of an Fc). Any of a variety of chemical modifications can be made by known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis with tunicamycin, etc. Furthermore, the antibody can contain one or more non-classical amino acids.

[0162] In some embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites can be made, e.g., by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0163] When the single domain antibodies provided herein are fused to an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally linked to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the binding molecules provided herein can be made in order to create variants with certain improved properties.

[0164] In other embodiments, when a single domain antibody provided herein is fused to an Fc region, an antibody variant provided herein can have a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. For example, the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, as measured by MALDI-TOF mass spectrometry, by comparing to the sum of all sugar structures attached to Asn 297 (e.g., complex, hybrid, and high mannose structures), as described in WO 2008 / 077546. 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 antibodies. Such fucosylation variants can have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 and US 2004 / 0093621. Examples of publications directed to “defucosylated” or “lacking fucose” 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; WO 2005 / 053742; WO 2002 / 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 afucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108; and WO 2004 / 056312, inter alia at Example 11); and knockout cell lines, such as CHO cells knocked out for the alpha-1,6-fucosyltransferase gene FUT8 (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0165] Binding molecules comprising a single domain antibody provided herein further provide bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to an Fc region is bisected by GlcNAc. Such variants can have reduced fucosylation and / or improved ADCC function. Examples of such variants are described in, e.g., WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such variants can have improved CDC function. Such variants are described in, e.g., WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0166] In molecules comprising a single domain antibody of the application and an Fc region, one or more amino acid modifications can be introduced into the Fc region, thereby generating an Fc region variant. An Fc region variant can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0167] In some embodiments, the present application encompasses variants that have some, but not all, effector functions, making them desirable candidates for use in applications where the half-life of the binding molecule in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm reduction / elimination of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the binding molecules lack FcyR binding (hence likely to lack ADCC activity), but retain FcRn binding ability. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods can be employed (see, e.g., ACTI TM Non-radioactive cytotoxicity assays (CellTechnology, Inc. Mountain View, CA; and CytoTox 96®; Promega, Madison, WI; see also, e.g., US Patent No. 5,200,184 and 5,350,924) can also be used. Alternatively, cell death can be assessed by measuring release of lactate dehydrogenase (LDH) (see, e.g., US Patent No. 5,942,401). Nonradioactive cytotoxicity assays (Promega, Madison, WI). Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl Acad. Sci. USA 95:652-656 (1998). Clq binding assays can also be carried out to demonstrate that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be carried out (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al. Blood 101 :1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / kidney half-life determinations can also be carried out using methods known in the art (see, e.g., Petkova, S.B. et al. Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0168] Binding molecules with reduced effector function include those in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 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 substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0169] Certain variants described have increased or decreased binding to FcRs. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0170] In some embodiments, variants comprise one or more amino acid substitutions in the Fc region that result in improved ADCC, e.g., substitutions at positions 298, 333, and / or 334 of an Fc region (EU numbering of residues). In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0171] Binding molecules with prolonged half-lives and improved 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. J. Immunol. 24:249 (1994)), are described in US 2005 / 0014934 Al (Hinton et al.). Those molecules comprise an Fc region with one or more substitutions therein that improve Fc region binding to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). See also, Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, for further examples of Fc region variants.

[0172] In some embodiments, it can be desirable to create cysteine engineered antibodies in which one or more residues of an antibody are substituted with cysteine residues. In some embodiments, the residues that are substituted are present in the accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as described further herein.

[0173] Replacement, Deletion, or Insertion

[0174] A variation can be a substitution, deletion, or insertion of one or more codons encoding a single domain antibody or polypeptide that results in an amino acid sequence change compared to the original antibody or polypeptide. Sites of interest for substitutional mutagenesis include the CDRs and FRs.

[0175] Amino acid substitutions can be one amino acid replaced by another amino acid with similar structural and / or chemical properties, e.g., leucine replaced by serine, e.g., as a result of a conservative amino acid substitution. Standard techniques known in the art can be used to introduce mutations in the nucleotide sequences encoding the molecules provided herein, including, e.g., site-directed mutagenesis and PCR-mediated mutagenesis that result in, e.g., amino acid substitutions. Insertions or deletions can optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion comprises fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In a particular embodiment, the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. Permitted variations can be determined by making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variant for the activity exhibited by the parent antibody.

[0176] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides of several amino acids, 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.

[0177] Single domain antibodies produced by conservative amino acid substitutions are included in the present disclosure. In a “conservative amino acid substitution,” an amino acid residue is replaced with an amino acid residue that has a side chain with a similar charge. As noted above, families of amino acid residues with side chains of similar charge have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. Conservative (e.g., within groups of amino acids with similar properties and / or side chains) substitutions can be made to maintain or alter properties. Exemplary substitutions are shown in Table 2 below.

[0178] Table 2. Amino acid substitutions

[0179]

[0180] Amino acids can be grouped according to similarities in their side chain properties (see, e.g., Lehninger, Principles of Biochemistry, 2nd ed., pp. 993-1025 (Worth Publishers, Inc., New York, 1975)): Biochemistry 73-75 (2nd ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, any cysteine residue not involved in maintaining the proper conformation of a single domain antibody also can be substituted, generally with another amino acid such as alanine or serine, so as to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Non-conservative substitutions will require the

[0181] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant will have some of the biological properties of the parent antibody (e.g., increased affinity) and / or will have substantially retained certain biological properties of the parent antibody. Exemplary substitutional variants are those produced by affinity maturation techniques as described herein. Briefly, one or more hypervariable region residues are mutated, and the variant antibodies displayed on phage, and screened for specific biological activity (e.g., binding affinity).

[0182] Changes (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. Such changes can be made in CDR “hotspots,” i.e., residues encoded by codons that are mutated at a 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 variant antibodies or fragments thereof being tested for binding affinity. For example, affinity maturation by construction and selection from secondary libraries has been described in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O’Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves CDR-directed approaches, in which a few CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, e.g., using alanine scanning mutagenesis or modeling. The following sections provide more detailed descriptions of affinity maturation.

[0183] In some embodiments, substitutions, insertions, or deletions can occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) can be made in CDRs that do not substantially reduce binding affinity. In some embodiments of the variant VHH sequences provided above, each CDR is unaltered, or contains no more than 1, 2, or 3 amino acid substitutions.

[0184] One method for identifying residues or regions in an antibody that can be targeted as sites for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells, Science, 244: 1081-1085 (1989). In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interactions of the antibody with antigen are affected. Further substitutions can be introduced at the amino acids locations showing functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0185] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a 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 insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0186] Mutations can be made using methods known in the art, such as oligonucleotide- mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, Biochem J. 237: 1-7 (1986); and Zoller et al., Nucl. Acids Res. 10: 6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34: 315-23 (1985)), or other known techniques can be performed on the cloned DNA to create single domain antibody variant DNA.

[0187] 5.2.4. In vitro affinity maturation

[0188] In some embodiments, antibody variants having improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be made by in vitro affinity maturation. Like natural selection, in vitro affinity maturation is based on the principles of mutation and selection. Antibodies are displayed on the surface of organisms (e.g., bacteriophage, bacteria, yeast, or mammalian cells) or in association (e.g., covalent or non-covalent) with their encoded mRNA or DNA. Affinity selection of the displayed antibodies allows isolation of the organisms or complexes that carry the genetic information encoding the antibodies. Two or three rounds of mutation and selection using display methods such as phage display often results in antibody fragments with affinities in the low nanomolar range. Affinity matured antibodies can have nanomolar or even picomolar affinities for the target antigen.

[0189] Phage display is a method widely used to display and select antibodies. Antibodies are displayed on the surface of Fd or M13 bacteriophage as a fusion to a bacteriophage coat protein. Selection involves exposure to antigen to allow the antibody displayed by the phage to bind its target, a process known as “panning”. Phage that bind to the antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, Methods. Mol. Biol. 178: 1-37 (2002); and Bradbury and Marks, J. Immunol. Methods 290: 29-49 (2004).

[0190] In a yeast display system (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997); and Chao et al., Nat. Protocols 1 :755-68 (2006)), antibodies can be fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall via a disulfide bond to Agalp. Displaying proteins via Aga2p projects the proteins away from the cell surface, thereby minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen libraries to select antibodies with improved affinity or stability. Binding to soluble antigens of interest is determined by labeling the yeast with biotinylated antigen and a secondary reagent conjugated to a fluorophore, such as streptavidin. Changes in surface expression of the antibody can be measured by immunofluorescent labeling of hemagglutinin or c-Myc epitope tags flanking single chain antibodies (e.g., scFv). Expression has been shown to correlate with the stability of the displayed protein, so antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). Another advantage of yeast display is that the displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cell, utilizing endoplasmic reticulum chaperones and quality control mechanisms. Once maturation is complete, antibodies can be conveniently "titrated" for affinity while displayed on the yeast surface, without the need to express and purify each clone. One theoretical limitation of yeast surface display is that the size of the functional library can be smaller than other display methods; however, a recent method uses the mating system of yeast cells to create a combinatorial diversity estimated at 10 14

[0191] ​In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in cell-free systems. A DNA library encoding a particular antibody library is genetically fused to a spacer sequence that lacks a stop codon. The spacer sequence remains attached to the peptidyl tRNA and occupies the ribosome tunnel upon translation, thereby allowing the protein of interest to protrude from the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to a surface-bound ligand, thereby allowing the antibody and its encoding mRNA to be isolated by affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed back to cDNA, which can then be mutagenized and used in the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34:el27 (2006)). In mRNA display, puromycin is used as a linker molecule to establish a covalent bond between the antibody and the mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0192] Since these methods are performed entirely in vitro, they have two major advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be easily introduced after each round of selection, e.g., by a non-proofreading polymerase, since no library needs to be transformed after any diversification step.

[0193] In some embodiments, a mammalian display system can be used.

[0194] Diversity can also be introduced into the CDRs of an antibody library either targeted or via random introduction. The former approach includes sequential targeting of all CDRs of an antibody via high- or low-level mutagenesis, or targeting of isolated somatic hypermutation hotspots (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)) or residues suspected to influence affinity for experimental or structural reasons. Diversity can also be introduced by replacing naturally diversified regions via DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into framework residues (see, e.g., Bond et al., J. Mol. Biol. 348:699-709 (2005)), employ loop deletions and insertions in CDRs, or use hybridization-based diversification (see, e.g., U.S. Patent Publication No. 2004 / 0005709). Additional methods of generating diversity in CDRs are disclosed, e.g., in U.S. Patent No. 7,985,840. Other methods useful for generating antibody libraries and / or antibody affinity maturation are disclosed, e.g., in U.S. Patent Nos. 8,685,897 and 8,603,930 and U.S. Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0195] Screening of libraries can be accomplished by various techniques known in the art. For example, single domain antibodies can be immobilized on a solid support, column, pin, or cellulose / poly(vinylidene difluoride) membrane / other filter, expressed on a host cell attached to an adsorption plate or used for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method of panning a display library.

[0196] For a review of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23:1105-16 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010); and references therein.

[0197] 5.2.5. Modifications of single domain antibodies

[0198] Covalent modifications of single domain antibodies are included within the scope of the present disclosure. Covalent modifications include reacting a target amino acid residue of a single domain antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the single domain antibody. Other modifications include deamidation of glutamine and asparagine residues to the corresponding glutamic and aspartic acid residues, respectively; hydroxylation of proline and lysine; hydroxyphosphorylation of serine or threonine residues; a-aminomethylation of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins:Structure and Molecular Properties 79-86 (1983)); acetylation of the N-terminal amine; and amidation of any C-terminal carboxylic group.

[0199] Other types of covalent modifications of single domain antibodies included within the scope of the present disclosure include altering the natural glycosylation pattern of the antibody or polypeptide as described above (see, e.g., Beck et al., Curr. Pharm. Biotechnol. 9:482-501 (2008); and Walsh, Drug Discov. Today 15:773-80 (2010)), and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in, e.g., U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. Single domain antibodies of the present disclosure that bind to BCMA can also be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to prolong half-life and / or confer known Fc- mediated effector functions.

[0200] Single chain antibodies of the present disclosure that bind to BCMA can also be modified to form chimeric molecules comprising a single chain antibody that binds to BCMA fused to another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or an Fc region of an IgG molecule (see, e.g., Aruffo, Curr. Opin. Immunol. 8: 97-102 (1996); and Chamow and Ashkenazi, Curr. Opin. Immunol. 11: 548- 55 (1999)). Single chain antibodies that bind to BCMA can also be used to generate chimeric antigen receptors (CARs) that bind to BCMA, as described in more detail below. Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)). Single chain antibodies that bind to BCMA can also be used to generate chimeric antigen receptors (CARs) that bind to BCMA, as described in more detail below.

[0201] Also provided herein are fusion proteins comprising a single chain antibody of the present disclosure that binds to BCMA and a heterologous polypeptide. In some embodiments, the heterologous polypeptide that is genetically fused or chemically conjugated to the antibody can be used to target the antibody to a cell having cell surface expression of BCMA.

[0202] Also provided herein are antibody panels that bind to the BCMA antigen. In particular embodiments, the antibody panels have different association rates, different dissociation rates, different affinities, and / or different specificities for the BCMA antigen. In some embodiments, the panels comprise or consist of about 10 to about 1000 or more antibodies. The antibody panels can be used, for example, in 96- or 384-well plates for assays such as ELISA.

[0203] 5.2.6. Preparation of single domain antibodies

[0204] Methods of preparing single domain antibodies have been described. See, e.g., Els Pardon et al., Nature Protocol, 9(3):674 (2014). Single domain antibodies (e.g., VHHs) can be obtained using methods known in the art, for example, by immunizing a Camelid (e.g., a camel or llama) and obtaining hybridomas therefrom, or by cloning a single domain antibody library using molecular biology techniques known in the art, followed by selection of individual clones of the unselected library by ELISA or by using phage display.

[0205] The single domain antibodies provided herein can be produced by culturing cells transformed or transfected with a vector containing a nucleic acid encoding the single domain antibody. Polynucleotide sequences encoding the polypeptide components of the antibodies of the disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from antibody-producing cells, such as hybridoma cells or B cells, and sequenced. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a host cell. A number of vectors are available and known in the art that can be used for the purposes of the present disclosure. The choice of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Suitable host cells for expression of the antibodies of the disclosure include prokaryotes, such as Archaebacteria and Eubacteria, including gram-positive and gram-negative organisms; eukaryotic microbes such as filamentous fungi or yeasts; invertebrate cells, such as insect or plant cells; and vertebrate cells, such as mammalian host cell lines. The host cells are transformed with the expression vectors described above and cultured in conventional nutrient media, modified as appropriate for inducible promoters, selection of transformants, or amplification of the genes encoding the desired sequences. The antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0206] Methods of antibody production, including vector construction, expression, and purification, are further described in Pluckthun et al., Methods Enzymol. 178: 468-493 (1989); Bird et al., Science 242: 423-426 (1988); Hardman et al., Bio / Technology 67: 76-82 (1988); and Ladner et al., U.S. Patent No. 5,648,263. Antibody Engineering:Producing antibodies in Escherichia coli:From PCR to fermentation 203-52 (edited by McCafferty et al., 1996); Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, Current Protocols in Protein Science (2009); Tachibana and Takekoshi, Production of Antibody FabFragments in Escherichia coli, Antibody Expression and Production (Edited by Al-Rubeai, 2011); and Therapeutic Monoclonal Antibodies:From Bench to Clinic (Edited by An, 2009).

[0207] Of course, alternative methods well known in the art are expected to be used to prepare anti-BCMA single-domain antibodies. For example, the appropriate amino acid sequence or a portion thereof can be produced by direct peptide synthesis using solid-phase techniques (see, for example, Stewart et al.). Solid-Phase Peptide Synthesis (1969); and Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963). In vitro protein synthesis can be performed using manual techniques or by automation. The individual parts of the anti-BCMA antibody can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-BCMA antibody. Alternatively, as disclosed, for example, in U.S. Patent Nos. 5,545,807 and 5,827,690, the antibody can be purified from the cells or body fluids, such as milk, of a transgenic animal engineered to express the antibody.

[0208] Specifically, the single-domain antibodies or other BCMA binding agents provided in this article can be generated through the following steps: immunization of llamas, single-B cell sorting, V gene extraction, cloning of the BCMA binding agent (e.g., VHH-Fc fusion), followed by small-scale expression and purification. Additional screening of BCMA-binding single-domain antibodies and other molecules can be performed, including selection based on ELISA positivity, BLI positivity, and K+. D One or more of the following criteria must be less than 100 nM. These selection criteria can be combined, as described in Section 6 below. Furthermore, the ability of individual VHH binders (and other molecules bound to BCMA) to bind to BCMA-expressing cells can be determined. Such assays can be performed on BCMA-expressing cells using FACS analysis and measurement of the mean fluorescence intensity (MFI) of the fluorescently labeled VHH molecules. Several aspects mentioned above will be described in more detail below.

[0209] Polyclonal antibodies

[0210] Polyclonal antibodies are typically raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Using bifunctional or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2or R 1 N = C = NR (where R and R 1 independently lower alkyl), the relevant antigen is conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Examples of adjuvants which can be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Immunization schedules can be selected by those of ordinary skill in the art without undue experimentation.

[0211] For example, animals are immunized against the antigen, immunogenic conjugate, or derivative by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1 / 5 to 1 / 10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneously or intraperitoneally at the multiple sites. Seven to fourteen days later the animals are bled and the serum is tested for antibody titer. Animals are boosted until the titer plateaus. The conjugates also can be made into a protein fusion with a protein that has an epitope tag which can be recognized with an antibody. In addition, aggregating agents such as alum are suitable for enhancing the immune response.

[0212] Monoclonal antibodies

[0213] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerization or amidation) that can be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0214] For example, the monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0215] In the hybridoma method, a suitable host animal is immunized by introduction of the protein or fragment thereof used to immunize to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing protein. 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).

[0216] The immunizing agent will typically include the antigenic protein or a fusion variant thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103. The immortalized cell line is typically a transformed mammalian cell. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a culture medium, such as HAT medium.

[0217] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The presence of monoclonal antibodies directed against the desired antigen can be determined by measuring the binding affinity of the antibodies in the culture medium of the hybridoma cells. Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)).

[0218] After the hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and / or avidity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, e.g., D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as tumors in an animal.

[0219] Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, e.g., protein A-agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0220] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 and those described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The 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 otherwise produce immunoglobulins, to obtain the synthesis of monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs. 130: 151-188 (1992).

[0221] In another 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). 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 provide alternative approaches for the isolation of monoclonal antibodies.

[0222] DNA can also be modified by substituting the coding sequence (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)) or by covalent attachment of all or part of the coding sequence to a non-immunoglobulin protein or polypeptide. Such non-immunoglobulin proteins can be substituted to produce chimeric diabodies that contain one antigen binding site specific for an antigen and another antigen binding site specific for a different antigen.

[0223] Chimeric or hybrid antibodies can also be prepared using known methods in synthetic protein chemistry, including those involving crosslinker agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0224] Recombination in prokaryotic cells

[0225] Standard recombinant techniques can be used to obtain polynucleic acid sequences encoding the antibodies of the present disclosure. The desired polynucleic acid sequence can be isolated from the cells producing the antibodies, such as hybridoma cells, and sequenced. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. Vector components generally include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

[0226] Generally, plasmid vectors containing replicon and control sequences are those that supply replication of the vector within a host microorganism, and are compatible with available transformation techniques. Examples of vectors containing these components generally are well known in the art. For example, the pBR322 plasmid is commonly used in E. coli and has different selection markers associated with its replication. The pBR322 plasmid contains multiple cloning sites, and is useful for cloning and amplifying DNA fragments. The pBR322 plasmid is a derivative of the pBR322 plasmid, and is useful for expressing proteins in E. coli. The pBR322 plasmid is a derivative of the pBR322 plasmid, and is useful for expressing proteins in E. coli.

[0227] In addition, bacteriophage vectors containing replicon and control sequences compatible with a host microorganism can be used to transform the host, as a transformation vector. For example, bacteriophage such as GEM TM -11 can be used to prepare a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392.

[0228] The expression vectors of the present application can comprise two or more promoter- cistron pairs, encoding each polypeptide component. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that modulates its expression. Prokaryotic promoters are generally classified as either inducible or constitutive. Inducible promoters are promoters that increase the level of transcription of the cistron under their control in response to changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

[0229] A large number of promoters recognized by a variety of potential host cells are well known. The promoter of choice can be operably linked to the target gene-encoding DNA of the application by removal of the promoter from its source DNA by restriction enzyme digestion and insertion of the isolated promoter sequence into the vector of the application. Both native promoter sequences and a number of heterologous promoters can be used to direct amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow greater transcription and higher yields of expressed target genes than the native target polypeptide promoter.

[0230] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the beta-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters (e.g., other known bacterial or bacteriophage promoters) that function in bacteria are also suitable. Their nucleotide sequences have been published, thus allowing for the facile insertion of a promoter into a vector molecule.

[0231] In one aspect, each cistron within the recombinant vector comprises a component of a secretion signal sequence that directs transmembrane translocation of the expressed polypeptide. Typically, the signal sequence can be a component of the vector, or it can be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for purposes of the present application should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that are not capable of recognizing and processing a native signal sequence of a heterologous polypeptide, the signal sequence can be replaced with a prokaryotic signal sequence selected from, for example, the group consisting of alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP.

[0232] In some embodiments, production of the antibodies according to the present disclosure can occur in the cytoplasm of the host cell, thus not requiring the presence of a secretion signal sequence within each cistron. Certain host strains (e.g., E. coli trxB- strains) provide cytoplasmic conditions that favor disulfide bond formation, allowing expressed protein subunits to fold and assemble correctly.

[0233] Prokaryotic host cells suitable for expression of the antibodies of the present disclosure include archaea 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), Enterobacter, 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 one embodiment, E. coli cells are used as hosts. 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 Accession No. 27,325) and its derivatives, including strain 33D3 (U.S. Patent No. 5,639,635) having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8A ompT A (nmpc-fepE) degP41 kan R Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative, and not limiting. Methods for constructing derivatives of any of the above bacteria with defined genotypes are known in the art and described, for example, in Bass et al., Proteins, 8:309-314 (1990). Suitable bacteria are generally needed for selection in view of replicability of replicons in bacterial cells. For example, E. coli, Serratia, or Salmonella species can be suitable as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to provide replicons.

[0234] Generally, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can be added to the cell culture as needed.

[0235] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the desired coding sequences. Transformation means introducing DNA into a prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Calcium treatment with calcium chloride is commonly used for bacterial cells with substantial cell wall barriers. Another transformation method employs polyethylene glycol / DMSO. Another technique used is electroporation.

[0236] Prokaryotic cells used to produce the antibodies of the present application are known in the art and are grown in culture media appropriate for the cultivation of the selected host cell. An example of a suitable culture medium includes Luria Broth (LB) plus necessary nutritional supplements. In some embodiments, the culture medium also contains a selection agent selected 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 the culture medium to allow the growth of cells expressing an ampicillin resistance gene.

[0237] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources can be introduced individually at the appropriate concentration, or as a mixture with another supplement or medium, such as a complex nitrogen source. Optionally, the culture medium can contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. The prokaryotic host cells are cultured at a suitable temperature and pH.

[0238] If an inducible promoter is used in the expression vectors of the present application, protein expression is induced under conditions appropriate for activation of the promoter. In one aspect of the present application, the PhoA promoter is used to control transcription of the polypeptide. Thus, the transformed host cells are cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods 263:133-147 (2002)). As is known in the art, a variety of other inducers can be used, depending on the vector construct employed.

[0239] The expressed antibodies of the present disclosure are secreted into the periplasm of the host cell and recovered from the periplasm. Protein recovery generally involves disruption of the microorganism, typically 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. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. Cells can be removed from the culture, and the culture supernatant filtered and concentrated for further purification of the produced protein. The expressed polypeptides can be further isolated and identified using well-known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0240] Alternatively, proteins are produced in large quantities by fermentation processes. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. To improve the yield and quality of the antibodies of the present disclosure, various fermentation conditions can be modified. For example, chaperone proteins have been shown to aid in the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. J BioChem 274: 19601-19605 (1999); U.S. Patent No. 6,083,715; U.S. Patent No. 6,027,888; Bothmann and Pluckthun, J. Biol. Chem. 275: 17100-17105 (2000); Ramm and Pluckthun, J. Biol. Chem. 275: 17106-17113 (2000); Arie et al., Mol. Microbiol. 39: 199-210 (2001).

[0241] To minimize proteolysis of expressed heterologous proteins, particularly those that are sensitive to proteolysis, certain host strains that lack proteolytic enzymes can be used in the present application, as described, for example, in U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996). E. coli strains that lack proteolytic enzymes and are transformed with plasmids that overexpress one or more chaperone proteins can be used as host cells in expression systems that encode the antibodies of the present application.

[0242] Antibodies produced herein can be further purified to obtain a substantially homogeneous preparation for further assays and uses. Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse 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. In some embodiments, Protein A, e.g., immobilized on a solid phase, can be used for immunoaffinity purification of the binding molecules of the present disclosure. The solid phase to which the Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled-pore glass or silica acid column. In some embodiments, the column has been coated with a reagent, e.g., glycerol, in an attempt to prevent nonspecific sticking of contaminants. The solid phase is then washed to remove contaminants that are not specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0243] Recombination in eukaryotic cells

[0244] For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.

[0245] Vectors for eukaryotic hosts can also be insertions of signal sequences or other polypeptides that have specific cleavage sites at the N-terminus of mature proteins or polypeptides. The signal sequence chosen preferably is one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase) and is optimal for the expression of the antibody of the application. In mammalian cell expression, mammalian signal sequences as well as viral secretory leader sequences, e.g., the herpes simplex gD signal, can be used. The DNA for such precursor regions is ligated in reading frame with DNA encoding an antibody of the application.

[0246] Generally, a replication origin component is not needed for mammalian expression vectors (the early SV40 origin is generally only used with an early promoter).

[0247] Expression and cloning vectors can contain a selection gene, also termed a selectable marker. The selection gene encodes a protein that confers on cells a resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, a nutritional requirement, e.g., biotin or thymidine, or a capacity to utilize a unique

[0248] One example of a selection scheme utilizes drugs to inhibit the growth of host cells. Those cells that have been successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs nmycin, mycophenolic acid and hygromycin.

[0249] Another example of a selectable marker applicable to mammalian cells is one that encodes a protein that facilitates the uptake of a selectable marker. For example, a gene conferring dihydrofolate reductase (DHFR) activity is used to transform cells, which are then cultured in the presence of methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is employed, an exemplary suitable host cell is a CHO cell line deficient in DHFR activity. Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) can be transformed with a DNA coding sequence for a polypeptide, wild-type DHFR protein, and another selectable marker (e.g., aminoglycoside 3'-phosphotransferase, APH) by growing the cells in media containing a selection agent (e.g., an aminoglycoside antibiotic) for the selectable marker.

[0250] Expression and cloning vectors usually contain a promoter that is recognized by a host organism and is operably linked to nucleic acid encoding a desired polypeptide sequence. Eukaryotic genes have a region rich in AT, located about 25 to 30 bases upstream from the site where transcription starts. Another sequence can be included, found about 70 to 80 bases upstream from the start of transcription of many genes. The 3' end of the majority of eukaryotic genes can be a signal for addition of a poly A tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0251] Transcription of the polypeptide from the vector in mammalian host cells can be controlled, for example, by promoters compatible with the host cell system and which can be obtained from viral genomes, such as polyoma, fowlpox, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40); heterologous mammalian promoters, such as the actin promoter or the immunoglobulin promoter; heat shock promoters.

[0252] Transcription of DNA encoding antibodies of the present disclosure 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, a -fetoprotein and insulin). Examples of enhancers are the SV40 enhancer, which is located in the region from base pairs 100-270, the cytomegalovirus early promoter enhancer, the mul timomah enhancer, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer can be spliced into the vector at a position 5' or 3' to the polypeptide-encoding sequence, but is preferably located at a site 5' from the transcription initiation site.

[0253] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing 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 nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA. One useful transcription termination component is the bovine growth hormone polyadenylation region.

[0254] Host cells suitable for cloning or expression of DNA in accordance with the present application include hyper eukaryotic cells as described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CVl line (COS cells) (ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3B, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRl cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0255] Host cells can be transformed with the above-described expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0256] Host cells used to produce the antibodies of the present application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Re. 30,985 can be used as culture media for the host cells. Any of these media can be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN® TM pharmaceuticals), trace elements (defined as inorganic compounds not falling into the other nutrient categories that are required in trace amounts, typically in the range of pmol per liter by the cells), and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations that would be known to those of skill in the art. The 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 the ordinarily skilled artisan.

[0257] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first treated with a protease inhibitor such as PMSF to inhibit proteolysis. The antibody can then be recovered from the medium either by using affinity ligand chromatography, such as protein A, protein G, or protein A / G columns, or by centrifugation.

[0258] Protein compositions prepared from cells can be purified using methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The matrix to which the affinity ligands are attached is typically agarose, but other matrices can also be used. Compared to agarose, mechanically stable matrices (such as glass with controlled pore size or poly(styrene-divinylbenzene)) allow for faster flow rates and shorter processing times. Other protein purification techniques can also be used, such as fractionation on ion-exchange columns, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, and heparin sepharose. TM Chromatography on anion or cation exchange resin (such as a polyaspartic acid column), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation can be performed, depending on the antibody to be recovered. After any preliminary purification step, the mixture containing the antibody of interest and contaminants can be subjected to low-pH hydrophobic interaction chromatography.

[0259] 5.2.7. Binding molecules containing single-domain antibodies

[0260] In another aspect, this document provides binding molecules comprising the single-domain antibody (e.g., the VHH domain targeting BCMA) provided herein. In addition to the chimeric antigen receptor (CAR) provided herein as described in Section 5.3 below, in some embodiments, the single-domain antibody targeting BCMA provided herein is part of other binding molecules. Exemplary binding molecules of this disclosure are described herein.

[0261] Fusion protein

[0262] In various embodiments, the single-domain antibody provided herein can be genetically fused or chemically conjugated with another agent, such as a protein-based entity. The single-domain antibody can be chemically conjugated with the agent, or otherwise non-covalently conjugated with it. The agent can be a peptide or antibody (or a fragment thereof).

[0263] Therefore, in some embodiments, this document provides single-domain antibodies (e.g., VHH domains) recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) with heterologous proteins or peptides (or fragments thereof, e.g., peptides with about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acids or more than 500 amino acids) and their uses. In particular, this document provides fusion proteins comprising antigen-binding fragments (e.g., CDR1, CDR2, and / or CDR3) of the single-domain antibodies provided herein and heterologous proteins, peptides, or polypeptides.

[0264] In addition, the antibodies provided herein can be fused to a marker or "tag" sequence, e.g., a peptide, to facilitate purification. In particular embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, a hemagglutinin ("HA") tag, and a "FLAG" tag.

[0265] Methods for fusing or conjugating moieties, including polypeptides, to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al., eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, Controlled Drug Delivery 623-53 (Robinson et al., eds., 2nded. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al., eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, Monoclonal Antibodies for Cancer Detection and Therapy 303-16 (Baldwin et al., eds., 1985); Thorpe et al., Immunol. Rev. 62:119-58 (1982); U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT Publication Nos. WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88:10535-39 (1991); Traunecker et al., Nature, 331:84-86 (1988); Zheng et al., J. Immunol. 154:5590-600 (1995); and Vil et al., Proc. Natl. Acad. Sci.USA 89: 11337-41 (1992)).

[0266] For example, the fusion proteins can be generated by gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of a single domain antibody as provided herein, including, for example, antibodies with higher affinity and lower off-rate (see, e.g., U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998)). The antibody or encoded antibody can be altered by random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. The polynucleotide encoding an antibody provided herein can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. of one or more heterologous molecules.

[0267] In some embodiments, a single domain antibody provided herein (e.g., a VHH domain) is conjugated to a second antibody to form an antibody heteroconjugate.

[0268] In various embodiments, a single domain antibody is genetically fused to an agent. Genetic fusion can be achieved by placing a linker (e.g., a polypeptide) between the single domain antibody and the agent. The linker can be a flexible linker.

[0269] In various embodiments, a single domain antibody is genetically conjugated to a therapeutic molecule, utilizing a hinge region to link the single domain antibody to the therapeutic molecule.

[0270] Also provided herein are methods of making the various fusion proteins provided herein. The various methods described in Section 5.2.6 above can also be used to make the fusion proteins provided herein.

[0271] In one embodiment, the fusion proteins provided herein are recombinantly expressed. Recombinant expression of the fusion proteins provided herein can require construction of an expression vector containing a polynucleotide that encodes the protein or fragment thereof. Once a polynucleotide encoding a protein provided herein or fragment thereof is obtained, a vector for production of the molecule can be generated using recombinant DNA technology well known in the art. Thus, described herein are methods for making a protein by expressing a polynucleotide containing a nucleotide sequence encoding. Expression vectors containing the coding sequences and appropriate transcriptional and translational control elements can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding a fusion protein provided herein or fragment thereof or CDRs operably linked to a promoter.

[0272] The expression vectors can be transferred into host cells by conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce the fusion proteins provided herein. Thus, also provided herein are host cells containing a polynucleotide encoding a fusion protein provided herein or fragment thereof operably linked to a heterologous promoter.

[0273] A variety of host expression vector systems can be used to express the fusion proteins provided herein. Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, and they also represent systems for production of the fusion proteins provided herein in situ, when cells transformed or transfected with the appropriate nucleotide coding sequences are used. These expression systems include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). Bacterial cells such as E. coli or eukaryotic cells, particularly those used for expression of intact recombinant antibody molecules, can be used to express the recombinant fusion proteins. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in conjunction with vectors such as the major intermediate early gene promoter element from human cytomegalovirus are an effective expression system for antibodies or variants thereof. In a particular embodiment, expression of the nucleotide sequences encoding the fusion proteins provided herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0274] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the expressed fusion protein. For example, when large quantities of the fusion protein are to be produced, for the generation of pharmaceutical compositions, vectors which direct the expression of high levels of fusion protein products are appropriate. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 12: 1791 (1983)), in which the coding sequence can be inserted in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol. Chem. 24:5503-5509 (1989)) and the like. The pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from dissolved cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0275] In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing fusion protein in infected hosts (see, e.g., Logan and Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals can also be required for translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences; and various upstream (e.g., the "Kozak" sequence) and downstream sequence motifs. The initiation codon must be

[0276] Furthermore, a host cell strain can be chosen for its ability to modulate expression of the inserted gene or genes on a desired, particular basis. Modulation of this sort can be accomplished by aptamer- mediated up- or down-regulation of a regulator gene, such as a repressor protein or activator protein, respectively, which then modulates expression of the inserted gene or genes. Alternatively, a host cell strain can be chosen that modulates the process of translation of the inserted gene or genes, the process of translation affecting the amount and type of protein ultimately produced. Still further, a host cell strain can be chosen for its ability to process and modify a protein, once it has been produced. Different host cells have different capabilities and therefore one can choose a host cell strain based on the potential of the cell to process and modify the protein as desired. For example, a mammalian host cell can be used to express a recombinant protein having fucose, a common carbohydrate modification found on the majority of human glycoproteins. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.

[0277] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express fusion proteins can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection agent, and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the fusion protein. Such engineered cell lines can be particularly useful in screening and evaluating compositions which interact directly or indirectly with the binding molecule.

[0278] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 :223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)) ; gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)) ; neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5): 155-215 (1993)) ; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology can be routinely used to select the desired recombinant clone(s) and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression Current Protocols in Molecular Biology , John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), all of which are herein incorporated by reference. Gene Transfer and Expression Current Protocols in Human Genetics

[0279] ​​Expression levels of the fusion protein can be increased by vector amplification (for review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing the fusion protein is amplifiable, increasing the amount of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the fusion protein gene, production of the fusion protein will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0280] Host cells can be co-transfected with the various expression vectors provided herein. The vectors can contain the same selectable markers, which enable the corresponding encoded polypeptides to be expressed equally. Alternatively, a single vector encoding and capable of expressing multiple polypeptides can be used. The coding sequences can comprise cDNA or genomic DNA.

[0281] Once a fusion protein provided herein has been produced by recombinant expression, it can be purified by any of the methods known in the art for purifying polypeptides, for example, by chromatography (e.g., ion exchange, affinity, particularly protein A post affinity for particular antigens, size column, and Kappa select affinity chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Furthermore, fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0282] Immunoconjugates

[0283] In some embodiments, the present disclosure also provides immunoconjugates comprising any of the antibodies (e.g., anti-BCMA single domain antibodies) described herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope.

[0284] In some embodiments, the 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 U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl); an auristatin, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); a dolastatin; a calicheamicin or a derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); 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. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane, such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; a trichothecene; and CC1065.

[0285] In some embodiments, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, phytolacaemia A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0286] 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. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and Lu. When the radioconjugate is used for detection, it can comprise a radioactive atom for scintigraphic studies, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0287] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), azinites (such as bis-(p-azidophenylthio) ethylenediamine), bis-diazonium

[0288] The linker can be a "cleavable linker" that facilitates release of the conjugate agent in the cell, although noncleavable linkers are also contemplated herein. Linkers for use in conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., a hydrazone linker), disulfide-containing linkers, peptidase-sensitive linkers (e.g., a peptide linker comprising amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linker, thioether linkers, or hydrophilic linkers designed to evade transporter-mediated multidrug resistance.

[0289] The immunoconjugates or ADCs herein contemplate, without limitation, 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 SVSS (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0290] In other embodiments, the antibodies provided herein are conjugated or recombinantly fused to, e.g., a diagnostic molecule. Such diagnostics and detection can be accomplished, e.g., by coupling the antibody to detectable substance, including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin, or avidin / biotin; fluorescent materials of various sort, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent materials, such as, but not limited to, 225Ac gamma emitter, Auger emitter, beta emitter, alpha emitter, or positron emitter radioisotopes.

[0291] 5.3. Chimeric Antigen Receptors

[0292] In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising a single domain antibody (e.g., VHH) that binds to BCMA provided herein. Exemplary CARs comprising VHH domains of the application (i.e., VHH-based CARs) are illustrated in Section 6 below.

[0293] In some embodiments, a chimeric antigen receptor (CAR) provided herein comprises a polypeptide comprising: (a) an extracellular antigen binding domain comprising one or more single domain antibodies (sdAb) that specifically bind to BCMA as provided herein and optionally one or more additional binding domains; (b) a transmembrane domain; and (c) an intracellular signaling domain. Each component and additional region is described in more detail below.

[0294] 5.3.1. Extracellular Antigen Binding Domain

[0295] The extracellular antigen binding domain of the CAR described herein comprises one or more (e.g., any of 1, 2, 3, 4, 5, 6, or more) single domain antibodies. The single domain antibodies can be fused to each other directly via a peptide bond or via a peptide linker.

[0296] Single domain antibodies

[0297] The CAR of the disclosure comprises an extracellular antigen binding domain comprising one or more single domain antibodies. The sdAbs can be of the same or different origin and of the same or different size. Exemplary sdAbs include, but are not limited to, a heavy chain variable domain from a heavy chain-only antibody (e.g., VHH or V NAR), naturally light chain-deficient binding molecules, single domains derived from conventional 4-chain antibodies (e.g., V H or V L ), humanized heavy chain-only antibodies, human single domain antibodies produced from transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. Any sdAb known in the art or developed from the present disclosure, including the single domain antibodies described above in the present disclosure, can be used to construct the CARs described herein. The sdAb can be derived from any species, including but not limited to mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. Single domain antibodies contemplated herein also include naturally occurring single domain antibody molecules from species other than camelids and sharks.

[0298] In some embodiments, the sdAb is derived from naturally occurring single domain antigen binding molecules, which are referred to as heavy chain antibodies without light chains (also referred to herein as "heavy chain-only antibodies"). For example, such single domain molecules are disclosed in WO 94 / 04678 and Hamers-Casterman, C. et al. Nature 363:446-448 (1993). For clarity, the variable domains derived from heavy chain molecules that naturally lack light chains are referred to herein as VHH to distinguish from the conventional V H Such VHH molecules can be derived from antibodies produced in camelid species such as camels, llamas, alpacas, dromedaries, vicunas, and guanacos. Other species outside of camelids can produce heavy chain molecules that naturally lack light chains, and such VHHs are within the scope of the present disclosure. In addition, humanized versions of VHHs, as well as other modifications and variants, are also contemplated and within the scope of the present disclosure.

[0299] VHH molecules from camelids are about 10 times smaller than IgG molecules. They are single polypeptides and can be very stable, resisting extreme pH and temperature conditions. In addition, they can resist the action of proteases, unlike conventional 4-chain antibodies. Furthermore, antibodies produced in camelids can recognize epitopes other than those recognized by antibodies produced in vitro by using antibody libraries or by immunizing mammals other than camelids (see, e.g., WO9749805). Thus, a multispecific or multivalent CAR comprising one or more VHH domains can interact with a target more efficiently than a multispecific or multivalent CAR comprising antigen binding fragments derived from conventional 4-chain antibodies. Since VHHs are known to bind into "unusual" epitopes (e.g., cavities or grooves), the affinity of a CAR comprising such VHHs can be more suitable for therapeutic treatment than conventional multispecific polypeptides.

[0300] In some embodiments, the sdAb is derived from the variable region of an immunoglobulin found in cartilaginous fish. For example, the sdAb can be derived from an immunoglobulin isotype found in shark serum called a novel antigen receptor (NAR). Methods of generating single domain molecules derived from the variable region of NAR ("IgNAR") are described in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0301] In some embodiments, the sdAb is recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or in vitro generated (e.g., by phage display selection). In some embodiments, the amino acid sequence of the framework region can be altered by "camelization" of particular amino acid residues in the framework region. Camelization refers to the replacement or substitution of one or more amino acid residues in the (naturally occurring) V H one or more amino acid residues in the amino acid sequence of the domain is replaced or substituted by one or more amino acid residues present at one or more corresponding positions in the VHH domain of a heavy chain antibody. This can be done in a manner known in the art, which will be clear to the skilled person. Such "camelization" substitutions are preferably inserted at amino acid positions which form the V H -V L interface and / or at amino acid positions present at said interface, and / or at so-called Camelidae hallmark residues, as defined herein (see e.g. WO 94 / 04678; Davies and Riechmann FEBS Letters 339:285-290 (1994); Davies and Riechmann, Protein Engineering 9(6):531-537 (1996); Riechmann, J. Mol. Biol. 259:957-969 (1996); and Riechmann and Muyldermans, J. Immunol. Meth. 231 :25-38 (1999)).

[0302] In some embodiments, the sdAb is a human single domain antibody produced by a transgenic mouse or rat expressing human heavy chain segments. See, e.g., US20090307787, U.S. Patent No. 8,754,287, US20150289489, US20100122358, and WO2004049794. In some embodiments, the sdAb is affinity matured.

[0303] In some embodiments, a naturally occurring VHH domain against a particular antigen or target can be obtained from a (naive or immunized) library of camelid VHH sequences. Such methods can or can not involve the use of the antigen or target, or at least a portion, fragment, antigenic determinant, or epitope thereof, to screen such libraries using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immunized) VHH libraries can be used, e.g., VHH libraries obtained from (naive or immunized) VHH libraries by, e.g., techniques described in WO 00 / 43507, e.g., random mutagenesis and / or CDR shuffling.

[0304] In some embodiments, single domain antibodies are generated from conventional four-chain antibodies. See, e.g., EP 0 368 684; Ward et al., Nature, 341(6242): 544-6 (1989); Holt et al., Trends Biotechnol., 21(11): 484-490 (2003); WO 06 / 030220; and WO 06 / 003388.

[0305] In some embodiments, an extracellular antigen binding domain provided herein comprises at least one binding domain, and the at least one binding domain comprises a single domain antibody that binds to BCMA as provided herein, e.g., an anti-BCMA single domain antibody described in Section 5.2, above.

[0306] In some embodiments, a CAR is provided herein, the CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising an anti-BCMA sdAb; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb is an anti-BCMA sdAb as described in Section 5.2, above, including, e.g., the VHH domains in Table 4 and those having one, two, or all three CDRs in any of the VHH domains in Table 4. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or humanized.

[0307] More specifically, in some embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising an anti-BCMA single domain antibody; (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: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3.

[0308] In other embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising an anti-BCMA single domain antibody; (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: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 72; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0309] In some embodiments, provided herein is a 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 the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In other embodiments, provided herein is a 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 an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0310] In other embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (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: 1; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the second anti-BCMA sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 72; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. The two VHH domains can be in any order in the extracellular domain, i.e., the first or second VHH domain can be at the N-terminus in the extracellular domain.

[0311] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 10.

[0312] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 11.

[0313] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 12.

[0314] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 13.

[0315] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 14.

[0316] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 15.

[0317] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 16.

[0318] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 8.

[0319] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 10.

[0320] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 11.

[0321] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 12.

[0322] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 13.

[0323] In some more specific embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 14.

[0324] In some more particular embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 15.

[0325] In some more particular embodiments, provided herein is a CAR comprising a polypeptide comprising: (a) an extracellular antigen-binding domain comprising at least two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 16.

[0326] In other embodiments, the extracellular antigen-binding domain further comprises one or more additional antigen binding domains. The one or more additional binding domains bind to one or more additional antigens, for example 1, 2, 3, 4 or more additional single domain antibody binding regions (sdAbs) that target one or more additional antigens.

[0327] In some embodiments, the additional antigen targeted by the CAR of the present disclosure is a cell surface molecule. Single domain antibodies can be selected to recognize antigens that serve as cell surface markers on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen. In some embodiments, the tumor antigen is associated with a B cell malignancy. Tumors express a variety of proteins that can serve as target antigens for an immune response, particularly a T cell mediated immune response. The antigen targeted by the CAR can be an antigen on a single diseased cell, or an antigen expressed on different cells that each contribute to the disease. The antigen targeted by the CAR can be directly or indirectly involved in the disease.

[0328] Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen to target will depend on the particular type of cancer to be treated. Exemplary tumor antigens include, but are not limited to, a glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, 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, prostanoid, 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.

[0329] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a variety of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gplOO in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation- associated molecules such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens are the oncofetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. In addition to BCMA, B-cell differentiation antigens such as CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.

[0330] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not occur on other cells in the body. TAAs are not unique to tumor cells but are also expressed on normal cells under conditions that do not induce an immune tolerant state to the antigen. Expression of the antigen on the tumor can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0331] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor suppressors, e.g., p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations; e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., Epstein Barr Virus antigens EBVA and human papilloma virus (HPV) antigens E6 and E7.

[0332] Other protein-based large antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0333] In some more particular embodiments, the one or more additional antigens are 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.

[0334] In some embodiments, the sdAb provided herein is camelid, chimeric, human, or humanized.

[0335] In addition to an antigen binding domain in the extracellular domain, the CARs provided herein can also comprise one or more of the following: a linker (e.g., a peptide linker), a transmembrane domain, a hinge region, a signal peptide, an intracellular signaling domain, a costimulatory signaling domain, each of which will be described in more detail below.

[0336] For example, in some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta. In some embodiments, the intracellular signaling domain comprises a chimeric signaling domain (“CMSD”), wherein the CMSD comprises a plurality of immunoreceptor tyrosine-based activation motifs (“CMSD ITAMs”) optionally connected by one or more linkers (“CMSD linkers”). In some embodiments, the CMSD comprises, from N-terminus to C-terminus: an optional N-terminal sequence-CD3 delta ITAM-an optional first CMSD linker-CD3 epsilon ITAM-an optional second CMSD linker-CD3 gamma ITAM-an optional third linker-DAP12 ITAM-an optional C-terminal sequence (e.g., ITAM010 provided herein). In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD137. In some embodiments, the BCMA CAR further comprises a hinge domain (e.g., a CD8a 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 (e.g., a CD8a signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus: a CD8a signal peptide, an extracellular antigen binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a costimulatory signaling domain derived from CD137, and a CMSD. In other embodiments, the polypeptide comprises, from N-terminus to C-terminus: a CD8a signal peptide, an extracellular antigen binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3 zeta. In some embodiments, the BCMA CAR is monospecific. In some embodiments, the BCMA CAR is monovalent. In some embodiments, the BCMA CAR is multispecific. In some embodiments, the BCMA CAR is multivalent.

[0337] peptide linkers

[0338] The various single domain antibodies in the multispecific or multivalent CARs described herein can be fused to one another via a peptide linker. In some embodiments, the single domain antibodies are fused directly to one another without any peptide linker. The peptide linkers connecting the different single domain antibodies (e.g., VHHs) can be the same or different. The different domains of the CAR can also be fused to one another via a peptide linker.

[0339] Each peptide linker in the CAR can have the same or different length and / or sequence, depending on the structural and / or functional features of the single domain antibodies and / or the individual domains. Each peptide linker can be independently selected and optimized. The length, degree of flexibility, and / or other properties of the peptide linkers used in the CAR can have some impact on properties including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. For example, a longer peptide linker can be selected to ensure that two adjacent domains do not interfere with one another sterically. In some embodiments, a short peptide linker can be disposed between a transmembrane domain and the intracellular signaling domain of the CAR. In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) such that the adjacent domains can move freely relative to one another. For example, a glycine-serine doublet can be a suitable peptide linker.

[0340] The peptide linker can have any suitable length. In some embodiments, the length of the peptide linker is at least about any of 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 some embodiments, the length of the peptide linker is no more than about 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 fewer amino acids. In some embodiments, the length of the peptide linker is about any of 1 amino acid to about 10 amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 30 amino acids, 5 amino acids to about 15 amino acids, 10 amino acids to about 25 amino acids, 5 amino acids to about 30 amino acids, 10 amino acids to about 30 amino acids, 30 amino acids to about 50 amino acids, 50 amino acids to about 100 amino acids, or 1 amino acid to about 100 amino acids.

[0341] Peptide linkers can have naturally occurring sequences, or non-naturally occurring sequences. For example, sequences derived from the hinge region of heavy chain-only antibodies can be used as linkers. See, e.g., WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n , glycine-serine polymers (including, e.g., (GS) n , (GSGGS) n , (GGGS) n , and (GGGGS) n where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers are listed in the table below.

[0342] Table 3. Exemplary peptide linkers

[0343]

[0344]

[0345] Other linkers known in the art, e.g., as described in WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465; Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990); and Bird et al., Science 242:423-426 (1988), each of which disclosures are incorporated herein by reference, can also be included in the CARs provided herein.

[0346] 5.3.2. Transmembrane domain

[0347] The CARs of the present disclosure comprise a transmembrane domain that can be fused directly or indirectly to the extracellular antigen binding domain. The transmembrane domain can be derived from a natural source or a synthetic source. As used herein, “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane domain suitable for use in the CARs described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0348] Transmembrane domains are classified according to the three-dimensional structure of the transmembrane domain. For example, a transmembrane domain can form an alpha helix, a complex of more than one alpha helix, a beta-barrel structure, or any other stable structure capable of spanning the cell phospholipid bilayer. In addition, transmembrane domains can also or alternatively be classified according to transmembrane domain topology, including the number of times the transmembrane domain passes through the membrane and the orientation of the protein. For example, a single-pass transmembrane protein passes through the cell membrane once, and a multiple-pass transmembrane protein passes through the cell membrane at least twice (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or more). Membrane proteins can be defined as Type I, Type II, or Type III, depending on the topology of their termini and one or more membrane-spanning segments relative to the inside and outside of the cell. Type I membrane proteins have a single transmembrane region, and are oriented such that the N-terminus of the protein is present on the extracellular side of the cell lipid bilayer, while the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is present on the extracellular side of the cell lipid bilayer, while the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple transmembrane regions, and can be further subdivided according to the number of membrane-spanning segments and the location of the N- and C-termini.

[0349] In some embodiments, the transmembrane domain of a CAR described herein is derived from a Type I single-pass transmembrane protein. In some embodiments, transmembrane domains from multiple-pass transmembrane proteins are also suitable for use in a CAR described herein. Multiple-pass transmembrane proteins can comprise complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helix or beta sheet structures. In some embodiments, the N-terminus and C-terminus of a multiple-pass transmembrane protein are present on opposite sides of the lipid bilayer, e.g., 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 extracellular side.

[0350] In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain selected from the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, BCMA, 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.

[0351] In some specific embodiments, the transmembrane domain is derived from CD8a. In some embodiments, the transmembrane domain is a transmembrane domain of CD8a comprising the amino acid sequence of SEQ ID NO: 19.

[0352] The transmembrane domain used in the CARs described herein can also comprise at least a portion of a synthetic non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least about 20 amino acids, such as at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, such as in U.S. Patent No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0353] The transmembrane domains provided herein can 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 can comprise three or more amino acids and, in some embodiments, facilitates the orientation of the transmembrane domain in a 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.

[0354] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the CARs provided herein comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine can be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises predominantly hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydrophilicity or hydrophobicity or hydrophilic character of a protein or protein segment can be assessed by any method known in the art, such as the Kyte and Doolittle hydrophilicity analysis.

[0355] 5.3.3. Intracellular signaling domain

[0356] The CARs of the disclosure comprise an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one normal effector function of the immune effector cell expressing the CAR. The term "effector function" refers to a particular function of a cell. For example, an effector function of a T cell can be cytolytic activity or helper activity including secretion of cytokines. The term "cytoplasmic signaling domain" thus refers to that portion of a protein which transduces the effector function signal and directs the cell to carry out the particular function. While it is possible to employ the entire cytoplasmic signaling domain, it is not necessary in many cases to use the entire chain. In terms of using a truncated portion of the cytoplasmic signaling domain, such truncated portions can be used in place of the entire chain so long as it transduces the effector function signal. The term cytoplasmic signaling sequence thus is intended to include any truncated portion of the cytoplasmic signaling domain sufficient to transduce the effector function signal.

[0357] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of the immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of the immune effector cell. A "primary intracellular signaling domain" refers to a cytoplasmic signal sequence that acts in a stimulatory manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. As used herein, "ITAM" is a conserved protein motif that is typically found in the tail of signaling molecules expressed in many immune cells. The motif can comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid, yielding the conserved motif YxxL / Ix(6-8)YxxL / I. The ITAM within the signaling molecule is important for signal transduction within the cell, mediated at least in part by phosphorylation of the tyrosine residues in the ITAM upon activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in the signaling pathway. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from CD3 zeta, FcR gamma (FCER1G), FcR beta (Fcs Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0358] In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3 zeta. In some embodiments, the primary intracellular signaling domain is the cytoplasmic signaling domain of wild-type CD3 zeta. In some embodiments, the primary intracellular signaling domain of CD3 zeta comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the primary intracellular signaling domain is wild-type CD3 zeta. In some embodiments, the primary intracellular signaling domain is a functional mutant of the cytoplasmic signaling domain of CD3 zeta containing one or more mutations, e.g., Q65K.

[0359] 5.3.3.1. Chimeric signaling domains

[0360] In some embodiments, the CAR of the present disclosure comprises a chimeric signaling domain (“CMSD”), as described in PCT / CN2020 / 112181 and PCT / CN2020 / 112182, incorporated by reference in their entireties. The CMSD described herein comprises an ITAM (also referred to herein as a “CMSD ITAM”) and an optional linker (also referred to herein as a “CMSD linker”), arranged in a configuration that is different from any naturally occurring ITAM-containing parent molecule. For example, in some embodiments, the CMSD comprises two or more ITAMs that are directly connected to each other. In some embodiments, the CMSD comprises ITAMs that are connected by one or more “heterogenous linkers,” that is, linker sequences that do not originate from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs originate (e.g., a G / S linker), or that originate from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs originate. In some embodiments, the CMSD comprises two or more (e.g., 2, 3, 4, or more) identical ITAMs. In some embodiments, at least two of the CMSD ITAMs are different from each other. In some embodiments, at least one of the CMSD ITAMs does not originate from CD3 zeta. In some embodiments, at least one of the CMSD ITAMs is not ITAM1 or ITAM2 of CD3 zeta. In some embodiments, the CMSD does not comprise CD3 zeta ITAM1 and / or CD3 zeta ITAM2. In some embodiments, at least one of the CMSD ITAMs is CD3 zeta ITAM3. In some embodiments, the CMSD does not comprise any ITAM from CD3 zeta. In some embodiments, at least two of the CMSD ITAMs originate from the same ITAM-containing parent molecule. In some embodiments, the CMSD comprises two or more (e.g., 2, 3, 4, or more) ITAMs, wherein at least two of the CMSD ITAMs each originate from a different ITAM-containing parent molecule. In some embodiments, at least one of the CMSD ITAMs originates from an ITAM-containing parent molecule selected from the group consisting of CD3 epsilon, CD3 delta, CD3 gamma, Ig alpha (CD79a), Ig beta (CD79b), Fc epsilon RI beta, Fc epsilon RI gamma, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, and Syk.

[0361] Thus, for example, in some embodiments, a CMSD comprises a plurality of ITAMs ("CMSD ITAMs") optionally connected by one or more linkers ("CMSD linkers"), wherein: (a) the plurality (e.g., 2, 3, 4, or more) of CMSD ITAMs are directly connected to one another; (b) the CMSD comprises two or more (e.g., 2, 3, 4, or more) CMSD ITAMs that are connected by one or more linkers that are not derived from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs are derived (e.g., a G / S linker); (c) the CMSD comprises one or more CMSD linkers that are derived from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs are derived; (d) the CMSD comprises two or more (e.g., 2, 3, 4, or more) of the same CMSD ITAM; (e) at least one of the CMSD ITAMs is not derived from CD3 zeta; (f) at least one of the CMSD ITAMs is not ITAM1 or ITAM2 of CD3 zeta; (g) the plurality of CMSD ITAMs are each derived from a different ITAM-containing parent molecule; and / or (h) at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3 epsilon, CD3 delta, CD3 gamma, Ig alpha (CD79a), Ig beta (CD79b), Fc epsilon RI beta, Fc epsilon RI gamma, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, and Syk.

[0362] In some embodiments, the CMSD has two or more of the above features. For example, in some embodiments, (a) the plurality (e.g., 2, 3, 4, or more) of CMSD ITAMs are directly connected to each other, and (d) the CMSD comprises two or more (e.g., 2, 3, 4, or more) of the same CMSD ITAM. In some embodiments, (b) the CMSD comprises two or more (e.g., 2, 3, 4, or more) of the CMSD ITAMs connected by one or more linkers (e.g., G / S linkers) that are not derived from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs is derived, and (d) the CMSD comprises two or more (e.g., 2, 3, 4, or more) of the same CMSD ITAM. In some embodiments, (c) the CMSD comprises one or more CMSD linkers that are derived from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs is derived, and (d) the CMSD comprises two or more (e.g., 2, 3, 4, or more) of the same CMSD ITAM. In some embodiments, (f) at least one of the CMSD ITAMs is not ITAM1 or ITAM2 of CD3 zeta, and (h) at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3 epsilon, CD3 delta, CD3 gamma, Ig alpha (CD79a), Ig beta (CD79b), Fc epsilon R1 beta, Fc epsilon R1 gamma, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, and moesin. In some embodiments, (b) the CMSD comprises two or more (e.g., 2, 3, 4, or more) of the CMSD ITAMs connected by one or more linkers (e.g., G / S linkers) that are not derived from the ITAM-containing parent molecule, and (f) at least one of the CMSD ITAMs is not ITAM1 or ITAM2 of CD3 zeta. In some embodiments, (b) the CMSD comprises two or more (e.g., 2, 3, 4, or more) of the CMSD ITAMs connected by one or more linkers (e.g., G / S linkers) that are not derived from the ITAM-containing parent molecule, and (h) at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3 epsilon, CD3 delta, CD3 gamma, Ig alpha (CD79a), Ig beta (CD79b), Fc epsilon R1 beta, Fc epsilon R1 gamma, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, and moesin.In some embodiments, (b) the CMSD comprises two or more (e.g., 2, 3, 4, or more) CMSD ITAMs that are linked by one or more linkers (e.g., G / S linkers) that are not derived from the ITAM-containing parent molecule, (d) the CMSD comprises two or more (e.g., 2, 3, 4, or more) identical CMSD ITAMs, and (h) at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3s, CD35, CD3y, Ig a (CD79a), Ig b (CD79b), Fc e RI b, Fc e RI g, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, and moesin. In some embodiments, (c) the CMSD comprises one or more CMSD linkers that are derived from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs is derived, and (e) at least one of the CMSD ITAMs is not derived from CD3 zeta.

[0363] In some embodiments, the ISD of a CAR described herein consists essentially of (e.g., consists of) a CMSD. In some embodiments, the ISD further comprises a costimulatory signaling domain (e.g., a 4-1BB or CD28 costimulatory signaling domain), which can be located N-terminal or C-terminal to the CMSD and linked to the CMSD by an optional linking peptide within the CMSD (e.g., linked by an optional CMSD N-terminal sequence or an optional CMSD C-terminal sequence).

[0364] A CMSD described herein can serve as the primary signaling domain in the ISD, which acts in a stimulatory manner to induce immune effector functions. For example, the effector function of a T cell can be cytolytic activity or helper activity including secretion of cytokines. As used herein, “ITAM” refers to a conserved protein motif that can be present in the tail of signaling molecules expressed in many immune cells (e.g., T cells). ITAMs are found in the cytoplasmic domains of many cell surface receptors (e.g., TCR complexes) or subunits associated with them and play an important regulatory role in signal transduction. Traditional CARs typically comprise a primary intracellular signaling domain (ISD) of CD3 zeta, which contains 3 ITAMs, CD3 zeta ITAM1, CD3 zeta ITAM2, and CD3 zeta ITAM3. In some embodiments, an ITAM described herein is naturally occurring, i.e., can be found in a naturally occurring ITAM-containing parent molecule. In some embodiments, an ITAM is further modified, e.g., by making one, two, or more amino acid substitutions relative to the naturally occurring ITAM.

[0365] ITAMs typically comprise two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acid residues, where each x is independently any amino acid residue, resulting in the consensus motif YxxL / I-x6-8-YxxL / I. In some embodiments, the ITAM contains a negatively charged amino acid (D / E) at the +2 position relative to the first ITAM tyrosine (Y), resulting in the consensus sequence D / E-x0-2-YxxL / I-x6-8-YxxL / I. Exemplary ITAM-containing signaling molecules include CD3s, CD35, CD3y, CD3z, Ig a (CD79a), Ig b (CD79b), Fc e RI b, Fc e RI g, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, and Syk, also referred to herein as “ITAM-containing parent molecules.” The ITAMs known to be present in ITAM-containing parent molecules participate in intracellular signal transduction upon ligand engagement, mediated at least in part by phosphorylation of tyrosine residues in the ITAMs upon activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in signaling pathways.

[0366] In some embodiments, the ITAM-containing parent molecule is CD3z. In some embodiments, the CD3z ISD comprises CD3z ITAM1, CD3z ITAM2, CD3z ITAM3, and a non-ITAM sequence N-terminal to CD3z ITAM1, C-terminal to CD3z ITAM3, and connecting the three ITAMs.

[0367] In some embodiments, the CMSD comprises a plurality of ITAMs, wherein at least two of the ITAMs are directly connected to one another. In some embodiments, the CMSD comprises a plurality of ITAMs, wherein at least two of the ITAMs are connected by a heterologous linker. In some embodiments, the CMSD further comprises an N-terminal sequence (also referred to herein as a “CMSD N-terminal sequence”) located N-terminal to the N-terminal most CMSD ITAM. In some embodiments, the CMSD further comprises a C-terminal sequence (also referred to herein as a “CMSD C-terminal sequence”) located C-terminal to the C-terminal most CMSD ITAM. In some embodiments, one or more of the linker, the N-terminal sequence, and / or the C-terminal sequence is / are about 1 to about 15 (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any range therebetween) amino acids in length. In some embodiments, the heterologous linker is a G / S linker. In some embodiments, the heterologous linker is derived from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs is derived.

[0368] In some embodiments, a 3-ITAM containing CMSD comprises, from N’ to C’: an optional CMSD N-terminal sequence - a first CMSD ITAM - an optional first CMSD linker - a second CMSD ITAM - an optional second CMSD linker - a third CMSD ITAM - an optional CMSD C-terminal sequence. In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - a CD3 zeta ITAM1 - an optional first CMSD linker - a CD3 zeta ITAM2 - an optional second CMSD linker - a CD3 zeta ITAM3 - an optional CMSD C-terminal sequence, wherein at least one of the first CMSD linker and the second CMSD linker is absent or is heterologous to CD3 zeta.

[0369] In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - a CD3 zeta ITAM1 - an optional first CMSD linker - a CD3 zeta ITAM1 - an optional second CMSD linker - a CD3 zeta ITAM1 - an optional CMSD C-terminal sequence, wherein the optional first CMSD linker and / or the second CMSD linker can be absent or have any linker sequence suitable for effector function signaling of the CMSD (e.g., the first CMSD linker can be the same as the CD3 zeta first linker, the second CMSD linker can be the same as the CD3 zeta second linker).

[0370] In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - a CD3 zeta ITAM2 - an optional first CMSD linker - a CD3 zeta ITAM2 - an optional second CMSD linker - a CD3 zeta ITAM2 - an optional CMSD C-terminal sequence, wherein the optional first CMSD linker and / or the second CMSD linker can be absent or have any linker sequence suitable for effector function signaling of the CMSD (e.g., the first CMSD linker can be the same as the CD3 zeta first linker, the second CMSD linker can be the same as the CD3 zeta second linker).

[0371] In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - a CD3 zeta ITAM3 - an optional first CMSD linker - a CD3 zeta ITAM3 - an optional second CMSD linker - a CD3 zeta ITAM3 - an optional CMSD C-terminal sequence, wherein the optional first CMSD linker and / or the second CMSD linker can be absent or have any linker sequence suitable for effector function signaling of the CMSD (e.g., the first CMSD linker can be the same as the CD3 zeta first linker, the second CMSD linker can be the same as the CD3 zeta second linker).

[0372] In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - CD3 zeta ITAM1 - an optional first CMSD linker - CD3 zeta ITAM3 - an optional second CMSD linker - CD3 zeta ITAM3 - an optional CMSD C-terminal sequence. In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - CD3 zeta ITAM2 - an optional first CMSD linker - CD3 zeta ITAM3 - an optional second CMSD linker - CD3 zeta ITAM3 - an optional CMSD C-terminal sequence. In some embodiments, a CMSD does not comprise any ITAM of CD3 zeta (e.g., ITAM1, ITAM2, or ITAM3). In some embodiments, a 3-ITAM containing CMSD comprises one or more (e.g., 1, 2, or 3) ITAMs derived from a non-CD3 zeta ITAM containing parent molecule (e.g., CD3 epsilon, CD3 delta, CD3 gamma, Ig alpha (CD79a), Ig beta (CD79b), Fc epsilon RI beta, Fc epsilon RI gamma, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, or moesin), and one or more optional linkers connecting them can be absent or have any linker sequence suitable for the effector function signaling of the CMSD (e.g., the first CMSD linker can be the same as the CD3 zeta first linker, the second CMSD linker can be the same as the CD3 zeta second linker, or a G / S linker).

[0373] Thus, in some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - CD3 epsilon ITAM - an optional first CMSD linker - CD3 epsilon ITAM - an optional second CMSD linker - CD3 epsilon ITAM - an optional CMSD C-terminal sequence.

[0374] In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - DAP12 ITAM - an optional first CMSD linker - DAP12 ITAM - an optional second CMSD linker - DAP12 ITAM - an optional CMSD C-terminal sequence.

[0375] In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - Ig alpha ITAM - an optional first CMSD linker - Ig alpha ITAM - an optional second CMSD linker - Ig alpha ITAM - an optional CMSD C-terminal sequence.

[0376] In some embodiments, a CMSD described herein comprises from N’ to C’: an optional CMSD N-terminal sequence - Igβ ITAM - an optional first CMSD linker - Igβ ITAM - an optional second CMSD linker - Igβ ITAM - an optional CMSD C-terminal sequence.

[0377] In some embodiments, a CMSD described herein comprises from N’ to C’: an optional CMSD N-terminal sequence - FcεRIγ ITAM - an optional first CMSD linker - FcεRIγ ITAM - an optional second CMSD linker - FcεRIγ ITAM - an optional CMSD C-terminal sequence.

[0378] In some embodiments, a CMSD described herein comprises from N’ to C’: a cytoplasmic CD3ζ N-terminal sequence - a first CMSD ITAM - a CD3ζ first linker - a second CMSD ITAM - a CD3ζ second linker - a third CMSD ITAM - a CD3ζ C-terminal sequence, where all non-ITAM sequences within the CMSD (the cytoplasmic CD3ζ N-terminal sequence, the CD3ζ first linker, the CD3ζ second linker, and the CD3ζ C-terminal sequence) are identical to and located in the same positions as the sequences in which they naturally occur in the parent CD3ζ ISD, such a CMSD is also referred to as a “CMSD comprising a non-ITAM CD3ζ ISD framework”. For a CMSD comprising a non-ITAM CD3ζ ISD framework, the first / second / third CMSD ITAM can be independently selected from the group consisting of CD3δ ITAM, CD3γ ITAM, CD3ζ ITAM1, CD3ζ ITAM2, CD3ζ ITAM3, DAP12 ITAM, Igα ITAM, Igβ ITAM, and FcεRIγ ITAM, except that the first CMSD ITAM is CD3ζ ITAM1, the second CMSD ITAM is CD3ζ ITAM2, and the third CMSD ITAM is CD3ζ ITAM3. For example, in some embodiments, a CMSD described herein comprises (e.g., consists of) from N’ to C’: a cytoplasmic CD3ζ N-terminal sequence - DAP12 ITAM - a CD3ζ first linker - DAP12 ITAM - a CD3ζ second linker - DAP12 ITAM - a CD3ζ C-terminal sequence. In some embodiments, a CMSD described herein comprises (e.g., consists of) from N’ to C’: a cytoplasmic CD3ζ N-terminal sequence - CD3γ ITAM - a CD3ζ first linker - CD3γ ITAM - a CD3ζ second linker - CD3γ ITAM - a CD3ζ C-terminal sequence.

[0379] In some embodiments, a 4-ITAM-containing CMSD comprises, from N’ to C’: an optional CMSD N-terminal sequence - a first CMSD ITAM - an optional first CMSD linker - a second CMSD ITAM - an optional second CMSD linker - a third CMSD ITAM - an optional third CMSD linker - a fourth CMSD ITAM - an optional CMSD C-terminal sequence. For 5-ITAM-containing, 6-ITAM-containing, etc. CMSDs, the same applies. For CMSDs comprising four or more (e.g., 4, 5 or more) ITAMs, because ITAM-containing parent molecules typically comprise 1 ITAM (e.g., non-CD3 zeta ITAM-containing molecules such as CD3 epsilon, CD3 delta, CD3 gamma, Ig alpha (CD79a), Ig beta (CD79b), Fc epsilon R1 beta, Fc epsilon R1 gamma, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, or moesin) or 3 ITAMs (e.g., CD3 zeta), at least one of the ITAMs in the CMSD will be different from one of the ITAM-containing parent molecules, or derived from a molecule different from the ITAM-containing parent molecule, or located at a position different from where the ITAM naturally occurs in the ITAM-containing parent molecule, and thus a CMSD comprising four or more (e.g., 4, 5 or more) ITAMs can comprise an ITAM derived from any of the ITAM-containing parent molecules described herein (e.g., CD3 zeta), an optional linker can be absent, a cytoplasmic non-ITAM sequence derived from the ITAM-containing parent molecule, or have a heterologous sequence from the ITAM-containing parent molecule (e.g., can be a G / S linker). In some embodiments, a CMSD described herein comprises, from N’ to C’: an optional CMSD N-terminal sequence - a CD3 delta ITAM - an optional first CMSD linker - a CD3 epsilon ITAM - an optional second CMSD linker - a CD3 gamma ITAM (- an optional third CMSD linker - a DAP12 ITAM - an optional CMSD C-terminal sequence. In some embodiments, one or more of the optional CMSD linkers, CMSD N-terminal sequence, and CMSD C-terminal sequence is derived from a cytoplasmic non-ITAM sequence of an ITAM-containing parent molecule. In some embodiments, a CMSD comprises the sequence of SEQ ID NO: 53 (also referred to herein as “ITAM010” or “ITAM010 construct”).

[0380] In some embodiments, the CMSD described herein does not bind to or bind to Nef protein is reduced. In some embodiments, the CMSD does not bind to Nef (e.g., wild-type Nef such as wild-type SIV Nef, or mutant Nef such as mutant SIV Nef). In some embodiments, the CMSD does not comprise CD3 zeta ITAM1 and CD3 zeta ITAM2. In some embodiments, the plurality of CMSD ITAMs is selected from CD3 zeta ITAM3, DAP12, CD3 epsilon, Ig alpha (CD79a), Ig beta (CD79b), or Fc epsilon RI gamma. In some embodiments, the ITAMs within the CMSD are all CD3 zeta ITAM3. In some embodiments, the ITAMs within the CMSD are all CD3 epsilon ITAMs. In some embodiments, the CMSD comprises 3 ITAMs that are DAP12 ITAM, CD3 epsilon ITAM, and CD3 zeta ITAM3. In some embodiments, the binding between Nef (e.g., wild-type Nef such as wild-type SIV Nef, or mutant Nef such as mutant SIV Nef) and the CMSD is at least about 3%, 5%, or 10% less (e.g., at least about any of 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% less) than the binding between Nef and an ITAM-containing parent molecule (e.g., CD3 zeta, CD3 epsilon). In some specific embodiments, the CAR provided herein comprising a humanized anti-BCMA sdAb provided herein comprises a CMSD comprising the amino acid sequence of SEQ ID NO: 53, and the CAR is expressed in engineered T cells expressing a Nef protein variant, e.g., a mutant Nef comprising the amino acid sequence of SEQ ID NO: 51 (mutant SIV Nef M116).

[0381] As discussed above, the CMSDs described herein can comprise one or more optional CMSD linkers, optional CMSD C-terminal sequences, and / or optional CMSD N-terminal sequences. In some embodiments, at least one of the one or more CMSD linkers, CMSD C-terminal sequences, and / or CMSD N-terminal sequences is derived from the ITAM-containing parent molecule, e.g., is a linker sequence in the ITAM-containing parent molecule. In some embodiments, the CMSD linker, CMSD C-terminal sequence, and / or CMSD N-terminal sequence is heterologous, i.e., it is not derived from the ITAM-containing parent molecule (e.g., a G / S linker), or is derived from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs is derived. In some embodiments, at least one of the one or more CMSD linkers, CMSD C-terminal sequences, and / or CMSD N-terminal sequences is heterologous to the ITAM-containing parent molecule, e.g., can comprise a sequence that is different from any portion of the ITAM-containing parent molecule (e.g., a G / S linker). In some embodiments, the CMSD comprises two or more heterologous CMSD linkers. In some embodiments, the two or more heterologous CMSD linkers are identical to each other. In some embodiments, at least two of the two or more (e.g., 2, 3, 4, or more) heterologous CMSD linkers are identical to each other. In some embodiments, all of the two or more heterologous CMSD linkers are different from each other. In some embodiments, at least one of the CMSD linker, CMSD C-terminal sequence, and / or CMSD N-terminal sequence is derived from CD3 zeta.

[0382] The linkers, C-terminal sequences, and N-terminal sequences within the CMSD can be of the same or different lengths and / or orders, depending on the structural and / or functional features of the CMSD. The CMSD linkers, CMSD C-terminal sequences, and CMSD N-terminal sequences can be independently selected and optimized. In some embodiments, a longer CMSD linker (e.g., a linker of at least about any of 5, 10, 15, 20, 25, or more amino acids in length) can be selected to ensure that two adjacent ITAMs do not sterically interfere with each other. In some embodiments, a longer CMSD N-terminal sequence (e.g., a CMSD N-terminal sequence of at least about any of 5, 10, 15, 20, 25, or more amino acids in length) is selected to provide sufficient space for a signal transduction molecule to bind to the most N-terminal ITAM. In some embodiments, one or more of the CMSD linkers, C-terminal CMSD sequences, and / or N-terminal CMSD sequences are no more than about any of 25, 20, 15, 10, 5, or 1 amino acids in length. The CMSD linker length can also be designed to be the same as the length of an endogenous linker that connects ITAMs within an ISD of an ITAM-containing parent molecule. The CMSD N-terminal sequence length can also be designed to be the same as the length of a cytoplasmic N-terminal sequence that is between the most N-terminal ITAM and the membrane of an ITAM-containing parent molecule.

[0383] In some embodiments, the CMSD linkers are flexible linkers (e.g., comprising flexible amino acid residues such as Gly and Ser, e.g., Gly-Ser doublets). In some embodiments, the CMSD linkers are G / S linkers. In some embodiments, the CMSD N-terminal sequences and / or CMSD C-terminal sequences are flexible (e.g., comprising flexible amino acid residues such as Gly and Ser, e.g., Gly-Ser doublets).

[0384] The one or more optional CMSD linker, CMSD N-terminal sequence, and / or CMSD C-terminal sequence can have any suitable length. In some embodiments, the CMSD linker, CMSD N-terminal sequence, and / or CMSD C-terminal sequence is independently no more than about any of 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids in length. In some embodiments, the one or more CMSD linker, N-terminal sequence, and / or C-terminal sequence is independently any of about 1 amino acid to about 10 amino acids, about 4 amino acids to about 6 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 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, or about 1 amino acid to about 15 amino acids in length. In some embodiments, the one or more CMSD linker, CMSD N-terminal sequence, and / or CMSD C-terminal sequence is about 1 amino acid to about 15 amino acids in length.

[0385] 5.3.4. Costimulatory signaling domains

[0386] Many immune effector cells require costimulation in addition to stimulation of antigen-specific signals to promote cell proliferation, differentiation, and survival, as well as to activate the effector functions of the cell. In some embodiments, the CAR comprises at least one costimulatory signaling domain. As used herein, the term “costimulatory signaling domain” refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function. The costimulatory signaling domains of the chimeric receptors described herein can be a cytoplasmic signaling domain from a costimulatory protein that transduces a signal and modulates an immune cell, such as a T cell, NK cell, macrophage, neutrophil, or eosinophil mediated response. The “costimulatory signaling domain” can be the cytoplasmic portion of a costimulatory molecule. The term “costimulatory molecule” refers to a cognate binding partner on an immune cell (e.g., a T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the immune cell, such as, but not limited to, proliferation and survival.

[0387] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., any of about 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more identical costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, e.g., any two or more costimulatory proteins described herein. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (e.g., a cytoplasmic signaling domain of CD3 zeta) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., a cytoplasmic signaling domain of CD3 zeta) are fused to one another via an optional peptide linker. The primary intracellular signaling domain and the one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (e.g., a cytoplasmic signaling domain of CD3 zeta). Multiple costimulatory signaling domains can provide additive or synergistic signaling.

[0388] 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 is suitable for use in the CARs described herein. The type of costimulatory signaling domain is selected based on factors such as the type of immune effector cell (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil) that will express the effector molecule and the desired immune effector function (e.g., ADCC effector).Examples of co-stimulatory signaling domains for CARs can be cytoplasmic signaling domains of co-stimulatory proteins including, but not limited to, 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 TNFRII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other co-stimulatory molecule, 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.

[0389] In some embodiments, the one or more co-stimulatory signaling domains are selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83 (e.g., CD83 and MD-2).

[0390] In some embodiments, the intracellular signaling domain in the CAR of the present disclosure comprises a co-stimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3 zeta and a co-stimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling domain of CD137 comprising the amino acid sequence of SEQ ID NO: 20.

[0391] Also within the scope of the present disclosure are variants of any of the co-stimulatory signaling domains described herein such that the co-stimulatory signaling domain is capable of modulating an immune response of an immune cell. In some embodiments, the co-stimulatory signaling domain comprises up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) as compared to the wild-type counterpart co-stimulatory signaling domain. Such a co-stimulatory signaling domain comprising one or more amino acid variations can be referred to as a variant. Mutation of an amino acid residue of a co-stimulatory signaling domain can result in increased signal transduction and enhanced stimulation of an immune response relative to a co-stimulatory signaling domain that does not comprise the mutation. Mutation of an amino acid residue of a co-stimulatory signaling domain can result in decreased signal transduction and attenuated stimulation of an immune response relative to a co-stimulatory signaling domain that does not comprise the mutation.

[0392] 5.3.5. Hinge Region

[0393] A CAR of the present disclosure can comprise a hinge domain positioned between the extracellular antigen binding domain and the transmembrane domain. A hinge domain is a segment of amino acids typically found between two domains of a protein and can allow for flexibility of the protein and movement of one or both domains relative to each other. Any amino acid sequence that provides for such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule can be used.

[0394] A hinge domain can contain about 10-100 amino acids, such as any of about 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, a hinge domain can be at least about any 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.

[0395] In some embodiments, a hinge domain is a hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art to comprise a hinge domain is suitable for use in the chimeric receptors described herein. In some embodiments, a hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to a chimeric receptor comprising the same. In some embodiments, a hinge domain is derived from CD8a. In some embodiments, a hinge domain is a portion of the hinge domain of CD8a, e.g., a fragment containing at least about 15 (e.g., any of 20, 25, 30, 35, or 40) contiguous amino acids of the hinge domain of CD8a. In some embodiments, the hinge domain of CD8a comprises the amino acid sequence of SEQ ID NO: 18.

[0396] A hinge domain of an antibody, such as an IgG, IgA, IgM, IgE, or IgD antibody, is also suitable for use in the pH-dependent chimeric receptor systems described herein. In some embodiments, a hinge domain is a hinge domain that connects the constant domains CH1 and CH2 of an antibody. In some embodiments, a hinge domain is of an antibody, and comprises the hinge domain of an antibody and one or more constant regions of an antibody. In some embodiments, a hinge domain comprises the hinge domain of an antibody and the CH3 constant region of an antibody. In some embodiments, a hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of an antibody. In some embodiments, an antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, an antibody is an IgG antibody. In some embodiments, an antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, a hinge region comprises the hinge region and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, a hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0397] Non-naturally occurring peptides can also be used as the hinge domain of the chimeric receptors described herein. In some embodiments, the hinge domain located between the C-terminus of the extracellular ligand binding domain of the Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, where x and n can independently be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0398] 5.3.6. Signal Peptide

[0399] The CARs of the disclosure can comprise a signal peptide (also known as a signal sequence) at the N-terminus of the polypeptide. In general, a signal peptide is a peptide sequence that targets the polypeptide to a desired site in the cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of the cell and will allow the effector molecule to integrate and anchor into the lipid bilayer. Those of skill in the art will readily appreciate signal peptides suitable for use in the CARs described herein, including signal sequences of naturally occurring proteins or synthetic non-naturally occurring signal sequences. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8a, GM-CSF receptor a, and IgGl heavy chain. In some embodiments, the signal peptide is derived from CD8a. In some embodiments, the signal peptide of CD8a comprises the amino acid sequence of SEQ ID NO: 17.

[0400] 5.3.7. Exemplary CARs

[0401] Exemplary CARs, such as those in Tables 5 and 6, including, for example, LIC948A22, LIC948A22H31, LIC948A22H32, LIC948A22H33, LIC948A22H34, LIC948A22H35, LIC948A22H36, LIC948A22H37, LUC948A22 UCAR, LUC948A22H34, LUC948A22H36, and LUC948A22H37, were generated as shown in Section 6 below.

[0402] In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 27. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 28. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 29. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 30. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 31. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 32. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 34.

[0403] In certain embodiments, the CAR provided herein comprises an amino acid sequence that is a certain percentage identical to any one of the CARs exemplified in Section 6 below.

[0404] In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 29.In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is a BCMA CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34.

[0405] In some embodiments, provided herein is an isolated nucleic acid encoding any of the BCMA CARs provided herein. A more detailed description of nucleic acid sequences and vectors is provided below.

[0406] 5.4. Engineered immune effector cells

[0407] In yet another aspect, provided herein is a host cell (e.g., an immune effector cell) comprising any of the CARs described herein.

[0408] Accordingly, in some embodiments, provided herein is an engineered immune effector cell (e.g., T cell) comprising a CAR, the CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising one or more anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb is an anti-BCMA sdAb as described in Section 5.2 above, including, for example, the VHH domains in Table 4 and those having one, two, or all three CDRs in any of those VHH domains in Table 4. In particular, the one or more anti-BCMA sdAbs are selected from: an anti-BCMA sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and an anti-BCMA sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 72, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-BCMA sdAb is camelid, chimeric, human, or humanized. In some embodiments, the transmembrane domain is selected from the group consisting of CD8a, 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 (e.g., T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta. In some embodiments, the primary intracellular signaling domain is a chimeric signaling domain (CMSD), e.g., ITAM010. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the CAR further comprises a hinge domain (e.g., a CD8a 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 CAR further comprises a signal peptide (e.g., a CD8a signal peptide) located N-terminal to the polypeptide. In some embodiments, the polypeptide comprises, from N- to C-terminus: a CD8a signal peptide, an extracellular antigen binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3 zeta.In other embodiments, the polypeptide comprises, from N- to C-terminus: a CD8a signal peptide, an extracellular antigen binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a costimulatory signaling domain derived from CD137, and a CMSD, e.g., ITAM010 provided herein.

[0409] In some embodiments, provided herein is an engineered immune effector cell (e.g., T cell) comprising a CAR, the CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising one or more anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NOs: 7-16. In some embodiments, provided herein is an engineered immune effector cell (e.g., T cell) comprising a CAR, the 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 an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NOs: 7-16. In specific embodiments, provided herein is an engineered immune effector cell (e.g., T cell) comprising a CAR, the CAR comprising a polypeptide comprising: (a) an extracellular antigen binding domain comprising two anti-BCMA sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein (1) the first anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 7 and the second anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 10; (2) the first anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 7 and the second anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 11; (3) the first anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 7 and the second anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 12; (4) the first anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 7 and the second anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 13; (5) the first anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 7 and the second anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 14; (6) the first anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 7 and the second anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 15; (7) the first anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 7 and the second anti-BCMA sdAb comprises an amino acid sequence of SEQ ID NO: 16.(8) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 8; (9) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 10; (10) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 11; (11) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 12; (12) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 13; (13) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 14; (14) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 15;or (15) the first anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the transmembrane domain is selected from the group consisting of CD8a, 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 (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3 zeta. In some embodiments, the primary intracellular signaling domain is a chimeric signaling domain (CMSD), e.g., ITAM010. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the CAR further comprises a hinge domain (e.g., a CD8a 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 CAR further comprises a signal peptide (e.g., a CD8a signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus: a CD8a signal peptide, an extracellular antigen binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3 zeta. In other embodiments, the polypeptide comprises, from N-terminus to C-terminus: a CD8a signal peptide, an extracellular antigen binding domain, a CD8a hinge domain, a CD8a transmembrane domain, a costimulatory signaling domain derived from CD137, and a CMSD, e.g., ITAM010 provided herein.

[0410] In other specific embodiments, provided herein is an engineered immune effector cell (e.g., a T cell) comprising a CAR comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 23-34, or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of SEQ ID NO: 23-34.

[0411] 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.

[0412] Also provided are engineered immune effector cells comprising (or expressing) two or more different CARs. Any two or more CARs described herein can be expressed in combination. The CARs can target different antigens, providing a synergistic or additive effect. The two or more CARs can be encoded on the same vector or on different vectors.

[0413] The engineered immune effector cells can also express one or more therapeutic proteins and / or immunomodulators, such as immune checkpoint inhibitors. See, e.g., International Patent Application Nos. PCT / CN2016 / 073489 and PCT / CN2016 / 087855, the entire contents of which are incorporated herein by reference.

[0414] 5.4.1. Vectors

[0415] The present disclosure provides vectors for cloning and expressing any of the CARs described herein. In some embodiments, the vector is suitable for replication and integration in 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 viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex viral vectors, 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), as well as other virology and molecular biology manuals.

[0416] Many viral-based systems have been developed for the transfer of genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A heterologous nucleic acid can be inserted into a vector and packaged in a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In some embodiments, a lentiviral vector is used. In some embodiments, a self-inactivating lentiviral vector is used. For example, a self-inactivating lentiviral vector carrying an immunomodulator (such as an immune checkpoint inhibitor) coding sequence and / or a self-inactivating lentiviral vector carrying a chimeric antigen receptor can be packaged using protocols known in the art. The resulting lentiviral vector can be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, as they allow long-term stable integration of the transgene and propagation in daughter cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0417] In some embodiments, a vector comprises any one of the nucleic acids encoding a CAR described herein. The nucleic acid can be cloned into a vector using any known molecular cloning methods 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. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present disclosure. Promoters can be broadly classified as constitutive promoters or regulated promoters, e.g., inducible promoters.

[0418] In some embodiments, the nucleic acid encoding the CAR is operably linked to a promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, a cytomegalovirus (CMV) promoter, a human elongation factor-1 alpha (hEF1a), a ubiquitin C promoter (UbiC), a phosphoglycerol kinase promoter (PGK), a simian virus 40 early promoter (SV40), and a chicken beta-actin promoter in combination with a CMV early enhancer (CAGG). The efficiency of such constitutive promoters in driving transgene expression has been extensively compared in numerous studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1a, UbiC, and PGK in driving chimeric antigen receptor expression in primary human T cells and concluded that the hEF1a promoter not only induced the highest levels of transgene expression, but also was best maintained in CD4 and CD8 human T cells (Molecular Therapy, 17(8): 1453-1464 (2009)). In some embodiments, the nucleic acid encoding the CAR is operably linked to an hEF1a promoter.

[0419] In some embodiments, the nucleic acid encoding the CAR is operably linked to an inducible promoter. Inducible promoters belong to the class of regulatory promoters. Inducible promoters can be induced by one or more conditions, such as a physical condition of the engineered immune effector cell, a microenvironment, or a physiological state of the engineered immune effector cell, an inducer (i.e., an inducible agent), or a combination thereof.

[0420] In some embodiments, the inducing condition does not induce expression of an endogenous gene in the engineered mammalian cell and / or in a subject receiving the pharmaceutical composition. In some embodiments, the inducing condition is selected from the group consisting of an inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of the engineered mammalian cell.

[0421] In some embodiments, the vector further contains a selectable marker gene or a reporter gene to select for cells expressing the CAR from a population of host cells transfected by the lentiviral vector. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to enable expression in the host cell. For example, the vector can contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the nucleic acid sequence.

[0422] In some embodiments, the vector comprises more than one nucleic acid encoding a CAR. In some embodiments, the vector comprises a nucleic acid containing 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 operatively linked to the second nucleic acid via 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.

[0423] 5.4.2. Immune effector cells

[0424] "Immune effector cells" are immune cells capable of performing immune effector functions. In some implementations, immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0425] 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, the T cells produce IL-2, TFN, and / or TNF after expressing a CAR and binding to target cells (such as BCMA+ tumor cells). In some embodiments, CD8+ T cells lyse antigen-specific target cells after expressing a CAR and binding to target cells.

[0426] In some embodiments, the immune effector cells are NK cells. In other embodiments, the immune effector cells may be established cell lines, such as NK-92 cells.

[0427] In some implementations, immune effector cells are differentiated from stem cells such as hematopoietic stem cells, pluripotent stem cells, iPS cells, or embryonic stem cells.

[0428] 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 the immune effector cells by transfecting any of the isolated nucleic acids or vectors described above. In some embodiments, the protein is inserted into the cell membrane while the cells are passed through a microfluidic system, such as a cell membrane. (See, for example, U.S. Patent Application Publication No. 20140287509) to introduce immune effector cells into CAR.

[0429] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The vectors can be transferred into immune effector cells by physical, chemical, or biological methods.

[0430] Physical methods of introducing a 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, e.g., 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.

[0431] Biological methods for introducing a 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 mammalian (e.g., human) cells.

[0432] Chemical methods of introducing a vector into an immune effector cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0433] In some embodiments, a RNA molecule encoding any of the CARs described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into an immune effector cell via known methods, e.g., mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17: 1027-1035 (2006).

[0434] In some embodiments, the transduced / transfected immune effector cell is propagated ex vivo following 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 any of 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 screened to select an engineered mammalian cell.

[0435] Reporter genes can be used to identify potential transfected cells and to assess the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and expression of the encoded polypeptide is manifested by some readily detectable property such as enzymatic activity. After DNA has been introduced into recipient cells, expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or a green fluorescent protein (GFP) 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 are commercially available.

[0436] Other methods of confirming the presence of a nucleic acid encoding a CAR ...

Claims

1. A chimeric antigen receptor (CAR), said CAR comprising a polypeptide including: (a) An extracellular antigen-binding domain comprising a first BCMA-binding portion and a second BCMA-binding portion, wherein the first BCMA-binding portion is a first anti-BCMA single-domain antibody and the second BCMA-binding portion is a second anti-BCMA sdAb; and wherein each of the first sdAb and the second sdAb is a VHH domain. (b) Transmembrane domains; and (c) Intracellular signal transduction domains, in: (i) The first anti-BCMA sdAb comprises CDR1 of the amino acid sequence shown in SEQ ID NO: 1; CDR2 of the amino acid sequence shown in SEQ ID NO: 2; and CDR3 of the amino acid sequence shown in SEQ ID NO: 3; and (ii) The second anti-BCMA sdAb comprises CDR1 of the amino acid sequence shown in SEQ ID NO: 4; CDR2 of the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 72; and CDR3 of the amino acid sequence shown in SEQ ID NO:

6.

2. The CAR of claim 1, wherein the first anti-BCMA sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9, and the second anti-BCMA sdAb comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

16.

3. The CAR as described in claim 2, wherein, (1) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 10; (2) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 11; (3) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 12; (4) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 13; (5) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 14; (6) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 15; (7) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 16; (8) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 8; (9) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 10; (10) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 11; (11) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 12; (12) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 13; (13) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 14; (14) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 15; (15) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 9, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 16; or (16) The first anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO: 7, and the second anti-BCMA sdAb contains the amino acid sequence of SEQ ID NO:

8.

4. The CAR as described in any one of claims 1 to 3, wherein the first anti-BCMA sdAb is at the N-terminus of the second anti-BCMA sdAb; or wherein the first anti-BCMA sdAb is at the C-terminus of the second anti-BCMA sdAb.

5. The CAR of any one of claims 1 to 3, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

6. The CAR of any one of claims 1 to 3, wherein the transmembrane domain is derived from CD8α or CD28.

7. The CAR of any one of claims 1 to 3, wherein the intracellular signal transduction domain comprises the major intracellular signal transduction domain of immune effector cells.

8. The CAR of claim 7, wherein the primary intracellular signal transduction domain originates from CD3ζ.

9. The CAR of any one of claims 1 to 3, wherein the intracellular signal transduction domain comprises a chimeric signal transduction domain ("CMSD").

10. The CAR of claim 9, wherein the CMSD comprises two, three, four or five immune receptor tyrosine-based activation motifs ("CMSD ITAM") connected via CMSD connectors.

11. The CAR of claim 9, wherein the CMSD comprises four immune receptor tyrosine-based activation motifs ("CMSD ITAM") linked by CMSD adapters, and the CMSD comprises, from N-terminus to C-terminus: N-terminal sequence - CD3δ ITAM - first CMSD adapter - CD3ε ITAM - second CMSD adapter - CD3γ ITAM - third adapter - DAP12 ITAM - C-terminal sequence.

12. The CAR of claim 9, wherein the CMSD comprises the amino acid sequence of SEQ ID NO:

53.

13. The CAR of any one of claims 1 to 3, wherein the intracellular signal transduction domain comprises a co-stimulatory signal transduction domain.

14. The CAR of claim 13, wherein the co-stimulatory signal transduction domain is derived from co-stimulatory molecules selected from the group consisting of ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

15. The CAR of claim 13, wherein the co-stimulatory signal transduction domain comprises a cytoplasmic domain of CD28 and / or a cytoplasmic domain of CD137.

16. The CAR of any one of claims 1 to 3, wherein the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

17. The CAR of claim 16, wherein the hinge domain is derived from CD8α.

18. The CAR of any one of claims 1 to 3, wherein the CAR further comprises a signal peptide located at the N-terminus of the polypeptide.

19. The CAR of claim 18, wherein the signal peptide is derived from CD8α.

20. A chimeric antigen receptor (CAR), said CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 23-34.

21. An isolated nucleic acid comprising a nucleic acid sequence encoding a CAR as described in any one of claims 1 to 14.

22. The isolated nucleic acid of claim 21, wherein the isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:35-46.

23. A vector comprising the isolated nucleic acid as described in claim 22.

24. An engineered immune effector cell, said engineered immune effector cell comprising a CAR as described in any one of claims 1-3, an isolated nucleic acid as described in claim 21 or claim 22, or a vector as described in claim 23.

25. The engineered immune effector cell of claim 24, wherein the immune effector cell is a T cell.

26. The engineered immune effector cell of claim 24, wherein the engineered immune effector cell further comprises exogenous Nef protein.

27. The engineered immune effector cell of claim 26, wherein the exogenous Nef protein is selected from the group consisting of SIV Nef, HIV1 Nef, HIV2 Nef and their subtypes.

28. The engineered immune effector cell of claim 26, wherein the exogenous Nef protein is wild-type Nef.

29. The engineered immune effector cell of claim 26, wherein the exogenous Nef protein is a mutant Nef, and the mutant Nef is a mutant SIV Nef containing the amino acid sequence of SEQ ID NO:

51.

30. A pharmaceutical composition comprising engineered immune effector cells and a pharmaceutically acceptable carrier as described in any one of claims 24 to 29.

31. Use of the engineered immune effector cells of any one of claims 24 to 29 or the pharmaceutical composition of claim 30 in the preparation of a medicament for treating a disease in a subject, wherein, The disease in question is multiple myeloma (MM).

Images