Multispecific antibodies targeting bcma

Abstract

A multispecific antibody targeting BCMA is provided. Specifically provided are multispecific antibodies, nucleic acids encoding them, vectors comprising the nucleic acids, cells comprising the vectors, pharmaceutical compositions comprising them, uses thereof in treating a subject having a BCMA expression-related disease are also provided.

Description

Technical Field

[0001] This invention relates to multispecific antibodies, and more particularly to multispecific antibodies targeting BCMA. Background Technology

[0002] B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRS17), is a protein encoded by the TNFRSF17 gene in the human body.

[0003] BAFF and APRIL are ligands of BCMA. BAFF (also known as BLYS, TALL-1, THANK, zTNF4, TNFSF20, D8Ertd387e) is a high-affinity ligand of BCMA, while APRIL (a proliferation-inducing ligand, also known as TNFSF13, TALL-2, TRDL-1) is a low-affinity ligand of BCMA. Furthermore, BAFF and APRIL are also ligands for B-cell activation factor receptor (BAFF-R), a member of the tumor necrosis factor receptor (TNFR) superfamily, as well as transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI). BCMA, along with BAFF-R and TACI, regulates B-cell proliferation, survival, maturation, and differentiation.

[0004] Multiple myeloma is a malignant plasma cell disease, and the expression level of BCMA is significantly increased in multiple myeloma cells. BCMA can be a suitable tumor antigen target for immunotherapeutic agents targeting multiple myeloma. Immunotherapy agents, such as antibodies that bind to BCMA, can block the binding between BCMA and its natural ligands BAFF and / or APRIL.

[0005] In camels, IgG antibodies, besides the traditional four-chain antibody IgG1 containing both light and heavy chains, also naturally exist as heavy-chain-only antibodies (HcAbs) IgG2 and IgG3, which do not contain a light chain. Heavy-chain-only antibodies contain a single variable domain (V... H H (or a single variable domain) possesses the characteristic of specifically binding to antigens and exhibits high affinity for them. Based on its uniqueness, V... H The H domain – alone or as part of a larger antigen-binding molecule – offers several significant advantages over conventional scFvs and antibody fragments such as Fab, including the ability to bind antigens with high affinity using only a single domain, and the capacity to be engineered into multivalent and multispecific forms. HThe H domain is highly soluble and has no tendency to aggregate; the molecules are small, thus exhibiting high tissue permeability; single-domain antibodies do not need to pair with light chains; and there is no light-heavy chain mismatch problem when forming multispecific antibodies, etc.

[0006] Multispecific antibodies targeting B cell maturation antigen (BCMA) and T cell CD3 receptor can redirect CD3 T cells to myeloma cells expressing BCMA to induce cytotoxic effects against the target cells.

[0007] As a potential therapeutic target, some antibodies targeting BCMA have been developed, but the options are still limited and more are needed. Summary of the Invention

[0008] This invention provides antibodies targeting BCMA, including multispecific antibodies targeting BCMA and activating T-cell antigens such as CD3. The invention also provides related nucleic acids, vectors, cells, compositions, preparation methods, and uses capable of encoding the provided antibodies.

[0009] On the one hand, the present invention provides a multispecific antibody comprising

[0010] (i) The first antigen binding module that binds to the first antigen;

[0011] (ii) the second antigen-binding module that binds to the second antigen; and,

[0012] (iii) The third antigen-binding module that binds to the first antigen;

[0013] Wherein the first antigen is BCMA and the second antigen is an activating T cell antigen, and both the first antigen-binding module and the third antigen-binding module are single variable domains and each independently contains any one of the following:

[0014] (a) CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9;

[0015] (b) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12;

[0016] (c) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15; or

[0017] (d) CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence shown in SEQ ID NO:18.

[0018] In some implementations, the first antigen-binding module and the third antigen-binding module each independently include any one of the following:

[0019] (b) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12; or

[0020] (c) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15.

[0021] In some embodiments, the first antigen-binding module comprises CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12; and the third antigen-binding module comprises CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15. Further, in some embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:42. In some specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39.

[0022] In other embodiments, the third antigen-binding module comprises CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12; and the first antigen-binding module comprises CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15. Further, in some embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:42, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:41. In some specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37.

[0023] In some implementations, the first antigen-binding module and the third antigen-binding module bind to different epitopes of BCMA.

[0024] The second antigen-binding module provides the ability to bind to activated T-cell antigens. In some embodiments, the second antigen-binding module includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the following complementarity-determining regions: HCDR1 containing the amino acid sequence shown in SEQ ID NO:43, HCDR2 containing the amino acid sequence shown in SEQ ID NO:44, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:45; the light chain variable region includes LCDR1 containing the amino acid sequence shown in SEQ ID NO:46, LCDR2 containing the amino acid sequence shown in SEQ ID NO:47, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:48. In a further specific embodiment, the heavy chain variable region of the second antigen-binding module contains the amino acid sequence shown in SEQ ID NO:49, and its light chain variable region contains the amino acid sequence shown in SEQ ID NO:50.

[0025] In some embodiments, the multispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.

[0026] In some specific embodiments, in the multispecific antibody, the first antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12; the third antigen-binding module comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15; and the second antigen-binding module comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following complementarity-determining regions: HCDR1 containing the amino acid sequence shown in SEQ ID NO:43, HCDR2 containing the amino acid sequence shown in SEQ ID NO:44, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:45; the light chain variable region comprises LCDR1 containing the amino acid sequence shown in SEQ ID NO:46, LCDR2 containing the amino acid sequence shown in SEQ ID NO:47, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:48. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:41, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:42, and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:49, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39, and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:49, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50. In these specific embodiments, in an optional configuration, both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is ScFv, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the second antigen-binding module. In a further, more specific embodiment, the multispecific antibody consists of two polypeptide chains, wherein the first polypeptide chain contains the aforementioned first antigen-binding module and one of the Fc polypeptides of the Fc domain, and the second polypeptide chain contains the aforementioned second antigen-binding module, third antigen-binding module and another Fc polypeptide of the Fc domain.

[0027] In some other specific embodiments, in the multispecific antibody, the third antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO:10, CDR2 comprising the amino acid sequence shown in SEQ ID NO:11, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:12; the first antigen-binding module comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO:13, CDR2 comprising the amino acid sequence shown in SEQ ID NO:14, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:15; and the second antigen-binding module comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following complementarity-determining regions: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:43, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:44, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:45; the light chain variable region comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:46, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:47, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:48. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:42, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:41, and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:49, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50. In a further specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37, and the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:49, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50. In these embodiments, an alternative configuration is provided in which both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module.In a further, more specific embodiment, the multispecific antibody consists of three polypeptide chains. The first polypeptide chain contains the aforementioned first antigen-binding module, third antigen-binding module, and one Fc polypeptide of the Fc domain. The second polypeptide chain contains the Fab heavy chain of the aforementioned second antigen-binding module and another Fc polypeptide of the Fc domain. The third polypeptide chain is the Fab light chain of the aforementioned second antigen-binding module. Alternatively, in another configuration, the first and third antigen-binding modules are both single variable domains. The second antigen-binding module is ScFv. The first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. In a further, more specific embodiment, the multispecific antibody consists of two polypeptide chains. The first polypeptide chain contains the aforementioned first antigen-binding module, third antigen-binding module, and one Fc polypeptide of the Fc domain. The second polypeptide chain contains the aforementioned second antigen-binding module and another Fc polypeptide of the Fc domain. In another alternative configuration, both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is a Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the Fab light chain of the second antigen-binding module. More specifically, in this embodiment, the multispecific antibody consists of three polypeptide chains: the first polypeptide chain contains the aforementioned first antigen-binding module and one of the Fc peptides in the Fc domain; the second polypeptide chain contains the aforementioned Fab heavy chain of the second antigen-binding module and another Fc peptide in the Fc domain; and the third polypeptide chain contains the aforementioned third antigen-binding module and the Fab light chain of the second antigen-binding module.

[0028] On the one hand, the present invention provides isolated nucleic acids comprising polynucleotides encoding the aforementioned multispecific antibodies.

[0029] On the one hand, the present invention provides a carrier containing the nucleic acid described in the present invention.

[0030] On the one hand, the present invention provides a host cell containing the nucleic acid or the vector described herein.

[0031] On the other hand, the present invention provides a method for preparing the multispecific antibody of the present invention, which includes culturing the host cells to express the multispecific antibody and isolating and purifying the multispecific antibody in the system.

[0032] On the other hand, the present invention provides a pharmaceutical composition comprising the aforementioned multispecific antibody and a pharmaceutically acceptable carrier.

[0033] On the other hand, the present invention provides the use of the multispecific antibody or the pharmaceutical composition thereof in the preparation of a medicament for treating BCMA expression-related diseases.

[0034] On the other hand, the present invention provides a method for treating BCMA expression-related diseases in subjects, comprising administering a therapeutically effective amount of the multispecific antibody or the pharmaceutical composition to the subjects.

[0035] In another aspect, the present invention provides an antibody that binds to BCMA, comprising a single variable domain, said single variable domain comprising:

[0036] (1) CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9;

[0037] (2) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12.

[0038] (3) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15; or

[0039] (4) CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence shown in SEQ ID NO:18.

[0040] In some embodiments, the single variable domain contains the amino acid sequence shown in SEQ ID NO:41 or 42, while the single variable domain does not contain the amino acid sequence shown in SEQ ID NO:21 or 23.

[0041] This invention provides multispecific antibodies targeting BCMA and activating T-cell antigens such as CD3. In some formulations, various forms of multispecific antibodies have been constructed using certain single variable domains, all exhibiting good anti-tumor effects, such as tumor cell lysis performance and safety. Attached Figure Description

[0042] Figure 1A For flow cytometry detection of anti-human BCMA V H Binding curve of H-Fc chimeric antibody to CHO-hBCMA cells;

[0043] Figure 1B For flow cytometry detection of anti-human BCMA V H Binding curve of H-Fc chimeric antibody with U266 cells;

[0044] Figure 1C For flow cytometry detection of anti-human BCMA V H Binding curve of H-Fc chimeric antibody to RPMI8226 cells;

[0045] Figure 1D For flow cytometry detection of anti-human BCMA V H Binding curve of H-Fc chimeric antibody to HUVEC cells;

[0046] Figure 2 To detect the binding curves of 1A10-Fc and 1A11-Fc chimeric antibodies with HEK293T-CynoBCMA or HEK293T by flow cytometry;

[0047] Figure 3 It showed anti-human BCMA V H ELISA results of competition between H-Fc chimeric antibody and ligand APRIL;

[0048] Figure 4A Comparative diagram of the extracellular amino acid sequences of BCMA in humans and cynomolgus monkeys;

[0049] Figure 4B The extracellular crystal structure of human BCMA;

[0050] Figure 5 This is a schematic diagram of the structure of an exemplary multispecific antibody of the present invention;

[0051] Figure 6 This is a schematic diagram of the structures of exemplary antibodies BC24, PC1, PC2, and PC3;

[0052] Figure 7 The binding curves of anti-BCMA / anti-CD3 multispecific antibodies to NCI-H929 cells;

[0053] Figure 8 The binding curves of anti-BCMA / anti-CD3 multispecific antibodies to MM1S cells;

[0054] Figure 9The binding curves of anti-BCMA / anti-CD3 multispecific antibodies to RPMI-8226 cells are shown.

[0055] Figure 10 For anti-BCMA / anti-CD3 multispecific antibodies and CD3 + T-cell binding curve;

[0056] Figure 11 The lysis rate of NCI-H929 tumor cells induced by effector cells of anti-BCMA / anti-CD3 multispecific antibodies at different concentrations;

[0057] Figure 12 The lysis rate of MM1S tumor cells induced by effector cells with different concentrations of anti-BCMA / anti-CD3 multispecific antibodies;

[0058] Figure 13 The lysis rate of RPMI-8226 tumor cells induced by effector cells of anti-BCMA / anti-CD3 multispecific antibodies at different concentrations;

[0059] Figure 14 The effect of anti-BCMA / anti-CD3 multispecific antibodies on T cell activation in the presence of target cells NCI-H929;

[0060] Figure 15 The effect of anti-BCMA / anti-CD3 multispecific antibodies on T cell activation in the presence of MM1S target cells;

[0061] Figure 16 The release levels of cytokines IL-2, IL-6, TNF-α, and IFN-γ were determined by co-incubating NCI-H929 cells and effector cells with different concentrations of anti-BCMA / anti-CD3 antibodies for 24 hours.

[0062] Figure 17 The effect of anti-BCMA / anti-CD3 antibodies on the growth of NCI-H929 subcutaneous xenografts;

[0063] Figure 18 The results of LC-MS analysis of the anti-BCMA / anti-CD3 antibody PC1 are shown. Invention Details

[0065] the term

[0066] The term "antibody" refers to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, etc.), and single-chain antibodies.

[0067] The term "multispecific" refers to an antibody's ability to specifically bind to multiple different antigenic determinants, such as two or more different antigenic determinants. Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different antigenic determinant. Different antigenic determinants can be expressed on the same or different cells. These different antigenic determinants can vary depending on the antigen (e.g., binding to antigens CD3 and BCMA) or can be present on the same antigen. An antigenic determinant is a specific chemical group with a certain composition and structure on the surface or other sites of an antigen molecule, capable of specifically binding to its corresponding antibody or sensitized lymphocyte. An example of an antigenic determinant is the B cell maturation antigen (BCMA), which has multiple antigenic determinants with defined or undefined structures. In this paper, any antibody that can bind to two or more different antigenic determinants on an antigen is called a multispecific antibody.

[0068] The term "N-valent antibody" indicates that the antibody has N antigen-binding sites. For example, a "bivalent antibody" means that the antibody has two antigen-binding sites, and a "trivalent antibody" means that the antibody has three antigen-binding sites. Natural human immunoglobulin molecules typically have two antigen-binding sites, Fab molecules typically have a single antigen-binding site, and single variable domain and ScFv molecules typically have a single antigen-binding site.

[0069] The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to an antigenic determinant. A specific antigen-binding module can be Fab, ScFv, or a single variable domain. In this article, antigenic determinant is synonymous with antigenic epitope.

[0070] The term "activating T-cell antigen" refers to an antigen expressed on the surface of T lymphocytes, such as cytotoxic T lymphocytes, which can induce T-cell activation upon interaction with an antibody. For example, the interaction between an antibody and an activating T-cell antigen can induce T-cell activation by triggering a signaling cascade of the T-cell receptor complex. In one specific embodiment, the activating T-cell antigen is CD3, for example, the ε subunit of CD3.

[0071] The term "T cell activation" refers to one or more cellular responses of T lymphocytes, such as cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, expression of activation markers, etc. The multispecific antibody of this invention can induce T cell activation. Suitable methods for measuring T cell activation are known in the art and described herein.

[0072] In this invention, the terms "first," "second," or "third" used to refer to antigen-binding modules, antigens, Fc peptides, peptide linkers, polypeptide chains, etc., are used for ease of distinction when more than one type of part is present. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific sequence or orientation to multispecific antibodies.

[0073] The term "fusion" refers to the connection of components (such as Fab, ScFv, Fc peptides, etc.) directly or via one or more peptide linkers through peptide bonds.

[0074] The term "variable domain" or "variable region" refers to a domain of an antibody involved in the binding of the antibody to an antigen. For example, natural four-chain antibodies (e.g., derived from humans, mice, etc.) have a heavy chain variable region (VH) and a light chain variable region (VL), while antibodies derived from animals such as camels or sharks have only a heavy chain variable domain. Each variable domain of a natural antibody is essentially composed of four "frame regions" and three "complementarity-determining regions." The four frame regions are referred to as frame region 1 (or FR1), frame region 2 (or FR2), frame region 3 (or FR3), and frame region 4 (or FR4), respectively; these frame regions are separated by three complementarity-determining regions (or CDRs) referred to in the art and hereinafter as complementarity-determining regions 1 (or CDR1), 2 (or CDR2), and 3 (or CDR3), respectively. Therefore, the general structure of a variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Variable domains endow antibodies with antigen specificity because they have antigen-binding sites.

[0075] The term "monovariable domain" refers to a variable domain capable of specifically binding to an antigenic epitope without pairing with other variable domains. A monovariable domain typically has three CDRs (CDR1, CDR2, and CDR3) located on a single domain. In some cases, the monovariable domain can be a heavy-chain variable domain (e.g., VH); as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit essentially composed of the monovariable domain, so that the monovariable domain does not need to interact with another variable domain to form a functional antigen-binding unit). Another example of a monovariable domain is the "VHH domain" (or simply "VHH" or "V") of the camelid family. H H”).

[0076] "CDR" (complementarity-determining region), also known as "hypervariant region (HVR)," generally refers to each region of an antibody variable region that is highly variable in sequence and / or forms a structurally defined loop. Naturally occurring four-chain antibodies typically contain six CDRs: three in the heavy chain variable region (HCDR1, HCDR2, and HCDR3) and three in the light chain variable region (LCDR1, LCDR2, and LCDR3). Heavy chain-only antibodies or single variable domain antibodies typically have three CDRs (CDR1, CDR2, and CDR3).

[0077] Currently, there are many methods for defining CDRs. The Kabat definition, based on sequence variability, is the most commonly used (Elvin A. Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); while the Chothia definition is based on the location of structural loops (Cyrus Chothia, et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901-917 (1987)). The AbM definition is a compromise between the Kabat and Chothia definitions and is used by the AbM antibody modeling software from Oxford Molecular. The "contact" definition of CDRs is based on the analysis of available complex crystal structures. However, it should be noted that the boundaries of CDRs for the same antibody variable region obtained by different methods of definition may differ; that is, the CDR sequences for the same antibody variable region defined by different methods may be different. Therefore, when referring to antibodies defined by a specific CDR sequence as defined by this invention, the scope of said antibodies also includes antibodies defined by CDR sequences converted to other arbitrary definitions (e.g., Chothia, AbM definitions, etc.).

[0078] The CDR sequences given in this article are generally classified according to the Kabat definition (Elvin A. Kabat, et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0079] The term “frame region” or “FR” refers to amino acid residues with variable domains other than the CDR residues defined herein.

[0080] The term "Fab" refers to a protein composed of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of an immunoglobulin. In this article, Fab refers to the Fab molecule in its native form, namely, the Fab heavy chain (VH-CH1, N-to-C-terminal direction) consisting of the variable region VH and the constant region CH1 of the heavy chain, and the Fab light chain (VL-CL, N-to-C-terminal direction) consisting of the variable region and the constant region CL of the light chain.

[0081] The term "scFv" encompasses the VH and VL domains of an immunoglobulin, wherein these domains are present within a single polypeptide chain. In some embodiments, the scFv also includes a peptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

[0082] The terms “Fc domain,” “Fc,” or “Fc region” are used herein to define the C-terminal region of the immunoglobulin heavy chain, which contains at least a portion of the constant region. This term includes both native sequence Fc and variant Fc. The C-terminal lysine residue (Lys447) of the Fc may or may not be present. Unless otherwise stated, the amino acid residues in the Fc or constant region are numbered according to the EU numbering system, also known as the EU index, described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIHP Publication 91-3242. As used herein, one “Fc polypeptide” of the Fc domain refers to one of the two polypeptides that form the dimer Fc domain. For example, the Fc polypeptide of the IgG Fc domain contains the IgG CH2 and IgG CH3 constant regions.

[0083] The term "effector function" refers to those biological activities attributable to the Fc domain of an antibody, which vary from antibody isotype to antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and so on.

[0084] The term "KD" as used herein refers to the dissociation constant, expressed as molar concentration (M). The KD value of an antibody can be determined using methods well-known in the art. For example, one method for determining antibody KD is using surface plasmon resonance, such as with biosensor systems like the Biacore system.

[0085] The term "treatment" refers to an attempt to alter the natural course of disease in an individual and can be a clinical intervention performed for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving the prognosis.

[0086] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Preferably, the subject according to the invention is a human. Unless otherwise stated, the terms "patient" and "subject" may be used interchangeably.

[0087] The term "isolated" means that the target compound (e.g., VHH, multispecific antibody, antibody or nucleic acid, etc.) has been isolated from its natural environment.

[0088] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the sequence to be aligned that are identical to those in the specific amino acid sequence shown herein, after aligning the sequence to be aligned with the specific amino acid sequence shown herein, and if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without any conserved substitutions as part of the sequence identity. Amino acid sequence identity alignment can be performed using various methods within the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the aligned sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0089] Various aspects of the present invention will be described in further detail in the following sections.

[0090] I. Single variable structure domain

[0091] This invention provides a single variable domain that binds to BCMA (such as human BCMA). The single variable domain provides more available options for the development or construction of drugs targeting BCMA. The single variable domain has good affinity for human BCMA, providing targeting specificity. In some cases, the single variable domain can provide the ability to block the binding of the proliferation-inducing ligand APRIL to BCMA. Specifically, in some embodiments, the single variable domain does not bind to human TACI and BAFF-R proteins, exhibiting specificity; in some embodiments, the single variable domain cross-reacts with monkey BCMA. Since monkeys are ideal experimental animals for drug toxicology experiments, cross-reactivity with monkeys will facilitate the conduct of drug toxicology studies.

[0092] This invention provides a single variable structural domain incorporating BCMA, comprising:

[0093] (1) CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9;

[0094] (2) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12.

[0095] (3) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15; or

[0096] (4) CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence shown in SEQ ID NO:18.

[0097] In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO:7, CDR2 of the amino acid sequence shown in SEQ ID NO:8, and CDR3 of the amino acid sequence shown in SEQ ID NO:9. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO:10, CDR2 of the amino acid sequence shown in SEQ ID NO:11, and CDR3 of the amino acid sequence shown in SEQ ID NO:12. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO:13, CDR2 of the amino acid sequence shown in SEQ ID NO:14, and CDR3 of the amino acid sequence shown in SEQ ID NO:15. In some embodiments, the single variable domain comprises: CDR1 of the amino acid sequence shown in SEQ ID NO:16, CDR2 of the amino acid sequence shown in SEQ ID NO:17, and CDR3 of the amino acid sequence shown in SEQ ID NO:18. In some embodiments, the single variable domain is of camel family. In some implementations, the single variable structural domain is humanized.

[0098] In some embodiments, the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, 40, 41, or 42. In some more specific embodiments, the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, and comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9. In some more specific embodiments, the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:41, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12. In some more specific embodiments, the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 37 or 38, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO: 10, CDR2 containing the amino acid sequence shown in SEQ ID NO: 11, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 12. In some more specific embodiments, the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:42, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15.In some more specific embodiments, the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:39 or 40, and comprises: CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15. In some more specific embodiments, the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:25, and comprises: CDR1 comprising the amino acid sequence shown in SEQ ID NO:16, CDR2 comprising the amino acid sequence shown in SEQ ID NO:17, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:18. In some embodiments, the amino acid sequence having at least 85%, 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 single variable domain comprising this sequence retains the ability to bind to BCMA. In some embodiments, a total of 1-18, 1-16, 1-14, 1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids are substituted, inserted, and / or deleted in the amino acid sequences selected from SEQ ID NO: 19, 41, 42, and 25. In some embodiments, the substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). In some embodiments, the substitution, insertion, or deletion occurs in the CDR region, for example, one, two, or three of CDR1, CDR2, and CDR3. In some embodiments, the substitution, insertion, or deletion occurs in both CDR and non-CDR regions.

[0099] In some embodiments, the amino acid sequence of the single variable domain comprises the amino acid sequence shown in SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, 40, 41 or 42.

[0100] In some embodiments, the amino acid sequence of the single variable domain is as shown in SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, 40, 41 or 42.

[0101] In some implementations, the single variable structural domain is V HH. In some implementations, V H H is humanized. A non-human single variable domain can be "humanized" by replacing one or more amino acid residues in the original single variable domain sequence with one or more amino acid residues present at the corresponding position in the human antibody VH domain. Humanization can be expected to reduce immunogenicity. Typically, the single variable domain has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0102] In some embodiments, the present invention provides a single variable domain that binds to the same epitope as any of the single variable domains described herein. In some specific embodiments, a single variable domain that binds to the same epitope as a single variable domain comprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, or 40 is provided. In some embodiments, the single variable domain binding to the same epitope is of cameloid origin or humanized.

[0103] Conventional techniques known to those skilled in the art can be used to competitively screen for binding to the same epitope. Therefore, in some embodiments, the present invention provides a single variable domain that competes with any of the single variable domains described herein for binding to BCMA. In some specific embodiments, a single variable domain that competes with a single variable domain containing the amino acid sequence SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, or 40 for binding to BCMA is provided. Binding to BCMA can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art. In some embodiments, the single variable domain that competes for binding to BCMA is of camel origin or humanized.

[0104] This invention provides several exemplary single variable domains that bind BCMA. The amino acid sequences of the exemplary single variable domain CDRs (CDR1, CDR2, and CDR3) provided by this invention are provided in Table S1 below. The full-length amino acid sequences of the exemplary single variable domains are provided in Table S2 below.

[0105] Table S1: CDR sequence of a single variable domain

[0106]

[0107] Table S2: Full-length sequence of single variable domains

[0108]

[0109]

[0110] SEQ ID NO:41 sequence:

[0111] QVQLVESGGGX1VQX2GGSLRLSCAASGYSSNVACMAWYRQAPGKX3X4EWVX5TIVADFGTTNYAASVKGRFTISQDNX6KNTVYLQMNSLX7X8EDX9AX 10 YYCAATQRGGIDWCDEINYWGQGTLVTVSS

[0112] Where X1 is S or L, X2 is P or A, X3 is G or E, X4 is R or L, X5 is A or S, X6 is A or S, X7 is R or K, X8 is A or P, X9 is T or S, X 10 It can be V or M.

[0113] SEQ ID NO:42 sequence:

[0114] QVQLVESGGGX 11 VQX 12 GGSLRLSCAASGX 13 TFNSACMGWFRQAPGKX 14 REGVX 15 RIETGYGGTVYADSVKGRFTISRDNX 16 KNTVYLQMNSLX 17 X 18 EDTAX 19 YYCAAKRSWCTPTWWHELDYNYWGQGTQVTVSS

[0115] Among them, X 11 For S or L, X 12 For P or A, X 13 For F or V, X 14 For E or G, X 15 For A or S, X 16 For A or S, X 17 For R or K, X 18 For A or P, X 19 It can be V or M.

[0116] The present invention provides an antibody that binds to BCMA and includes the aforementioned single variable domain.

[0117] In some implementations, the antibody that binds to BCMA may be a monospecific antibody or a multispecific antibody.

[0118] In one embodiment, the monospecific antibody comprises an Fc domain, preferably the Fc of human IgG1, IgG2, IgG3 or IgG4.

[0119] In some embodiments, the monospecific antibody comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:27, 29, 31, 33, 51, 53, 55, or 57.

[0120] The present invention provides exemplary monospecific antibodies (e.g., 1A10-V1, 1A10-V2, 1A11-V1, 1A11-V2, etc.) that fuse a single variable domain with the Fc of human IgG1 to form a homodimer through the Fc.

[0121] The present invention provides isolated nucleic acids comprising polynucleotides encoding the BCMA-binding antibody described in this invention.

[0122] The present invention provides a vector containing the nucleic acid described herein.

[0123] The present invention provides isolated host cells containing the nucleic acids or vectors described herein.

[0124] The present invention provides a method for preparing the BCMA-binding antibody of the present invention, comprising culturing the host cells of the present invention to express the BCMA-binding antibody, and isolating and purifying the BCMA-binding antibody in the system.

[0125] The present invention provides a pharmaceutical composition comprising an antibody that binds to BCMA as described herein and a pharmaceutically acceptable carrier.

[0126] The present invention provides a method for treating BCMA expression-related diseases in subjects, comprising administering to the subjects a therapeutically effective amount of the BCMA-binding antibody or the pharmaceutical composition described herein.

[0127] II. Multispecific antibodies

[0128] This invention provides a multispecific antibody containing a single variable domain, which can target BCMA expressed on the surface of tumor cells and can also bind to T cells to activate the tumor-killing activity of T cells.

[0129] Using a single variable structural domain, compared to using more (e.g., two) Fab structures, can reduce or avoid mismatches between light and heavy chains.

[0130] The multispecific antibodies of this invention also exhibit excellent antitumor properties, such as lysing tumor cells. In some embodiments, the multispecific antibodies of this invention can induce the killing of various tumor cells, and they demonstrate good tumor-killing activity against tumor cells with different BCMA expression levels. In particular, they exhibit superior killing performance against some tumor cells with low BCMA expression.

[0131] The multispecific antibody of the present invention exhibits good anti-tumor performance while having a low level of cytokine release, which can reduce the risk of cytokine storm caused by antibodies targeting CD3 and improve safety.

[0132] Multispecific antibodies can be constructed using any of the single variable domains described in Section I. Therefore, this invention provides multispecific antibodies comprising...

[0133] (i) The first antigen binding module that binds to the first antigen;

[0134] (ii) the second antigen-binding module that binds to the second antigen; and,

[0135] (iii) The third antigen-binding module that binds to the first antigen;

[0136] Wherein the first antigen is BCMA and the second antigen is an activating T cell antigen, and both the first antigen-binding module and the third antigen-binding module are single variable domains and each independently contains any one of the following:

[0137] (a) CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9;

[0138] (b) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12;

[0139] (c) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15; or

[0140] (d) CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence shown in SEQ ID NO:18.

[0141] In this invention, both the first antigen-binding module and the third antigen-binding module are single variable domains that bind BCMA. In some embodiments, the first antigen-binding module includes the complementarity-determining region described in (a), (b), (c), or (d) above. In some embodiments, the third antigen-binding module includes the complementarity-determining region described in (a), (b), (c), or (d) above. In the multispecific antibody, the first antigen-binding module and the third antigen-binding module may be the same or different, and any single variable domain described in this invention can be independently selected for combination; in one specific embodiment, the first antigen-binding module and the third antigen-binding module are the same; in another specific embodiment, the first antigen-binding module and the third antigen-binding module are different.

[0142] Table S3 exemplifies multispecific antibodies constructed using different combinations of the first antigen-binding module and the third antigen-binding module that define the CDR characteristics. In one specific embodiment, the first antigen-binding module includes the complementarity-determining region described in (b) above, and the third antigen-binding module includes the complementarity-determining region described in (c) above. Specifically, the first antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12; and the third antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15. In another specific embodiment, the third antigen-binding module includes the complementarity-determining region described in (b) above, and the first antigen-binding module includes the complementarity-determining region described in (c) above. Specifically, the third antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12; and the first antigen-binding module includes: CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15. More specifically, the first antigen-binding module includes CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12; and the third antigen-binding module includes CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15. In another more specific embodiment, the third antigen-binding module comprises CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12; and the first antigen-binding module comprises CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15.

[0143] Table S3 provides exemplary multispecific antibodies that define the CDR characteristics of the first and third antigen-binding modules.

[0144]

[0145] Note: In the table, (X)+(Y) represents the combination of the third antigen-binding module and the first antigen-binding module in a multispecific antibody. For example, (b)+(a) means that the third antigen-binding module in a multispecific antibody includes the complementarity-determining region described in (b), while the first antigen-binding module includes the complementarity-determining region described in (a).

[0146] Furthermore, in some embodiments, the first antigen-binding module and the third antigen-binding module each independently comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, 41, 42, or 25.

[0147] In some embodiments, the first antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, 41, 42, or 25. In some embodiments, the first antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, 41, 42, or 25. In some specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 19, 41, 42, or 25. In some more specific embodiments, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 19, 21, 23, 37, 38, 39, 40 or 25.

[0148] In some embodiments, the third antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, 41, 42, or 25. In some embodiments, the third antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, 41, 42, or 25. In some specific embodiments, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 19, 41, 42, or 25. In some more specific embodiments, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 19, 21, 23, 37, 38, 39, 40 or 25.

[0149] In some specific embodiments, the first antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:41; and the third antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:42. In one specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:41, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:42. In some more specific embodiments, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:21, 37 or 38, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:23, 39 or 40.

[0150] In some embodiments, the first antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:42, and the third antigen-binding module comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:41. In one specific embodiment, the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:42, and the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:41. In some more specific embodiments, the first antigen-binding module comprises...

[0151] The third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:23, 39 or 40, and the amino acid sequence shown in SEQ ID NO:21, 37 or 38.

[0152] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:21, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:21, or SEQ ID NO:40.

[0153] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:21, and the first antigen-binding module comprises the amino acid sequence shown in 21, 23, 37, 38, 39, or 40.

[0154] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:37, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:39.

[0155] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:37, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:38.

[0156] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:37, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:40.

[0157] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:38, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:39.

[0158] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:38, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:40.

[0159] In one example, the first antigen-binding module contains the amino acid sequence shown in SEQ ID NO:39, and the third antigen-binding module contains the amino acid sequence shown in SEQ ID NO:40.

[0160] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39.

[0161] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:38.

[0162] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:40.

[0163] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:38, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39.

[0164] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:38, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:40.

[0165] In one example, the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:40.

[0166] In one example, both the first antigen-binding module and the third antigen-binding module contain the amino acid sequence shown in SEQ ID NO: 21, 23, 37, 38, 39 or 40.

[0167] Table S4 illustrates multispecific antibodies constructed using different combinations of the first and third antigen-binding modules, which feature the full-length amino acid sequence.

[0168] Table S4 provides examples of multispecific antibodies that define the amino acid sequences of the first and third antigen-binding modules.

[0169]

[0170]

[0171] Note: In the table, (X)+(Y) represents the combination of the third antigen-binding module and the first antigen-binding module in a multispecific antibody. For example, "1A10+1A1 single variable domain" means that the third antigen-binding module in a multispecific antibody is the amino acid sequence of the 1A10 single variable domain, and the first antigen-binding module is the amino acid sequence of the 1A1 single variable domain. The amino acid sequences of the single variable domains involved in this table are shown in Table S2.

[0172] In this invention, the first antigen-binding module can be from a camel or humanized. The third antigen-binding module can be from a camel or humanized. Humanization can reduce immunogenicity; in some embodiments, both the first and third antigen-binding modules are humanized. In some embodiments, both the first and third antigen-binding modules are from camels.

[0173] In the multispecific antibody of the present invention, the second antigen-binding module provides the ability to target activating T-cell antigens. The second antigen-binding module can be Fab, ScFv, or ScFab. In one embodiment, the activating T-cell antigen is CD3. In another specific embodiment, the second antigen-binding module is a Fab that binds to CD3. In yet another specific embodiment, the second antigen-binding module is a ScFv that binds to CD3.

[0174] In some implementations, the second antigen-binding module is murine, chimeric, or humanized.

[0175] In some embodiments, the second antigen-binding module includes a heavy chain variable region and a light chain variable region, the heavy chain variable region including the following complementarity-determining regions: HCDR1 containing the amino acid sequence shown in SEQ ID NO:43, HCDR2 containing the amino acid sequence shown in SEQ ID NO:44, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:45; the light chain variable region including LCDR1 containing the amino acid sequence shown in SEQ ID NO:46, LCDR2 containing the amino acid sequence shown in SEQ ID NO:47, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:48.

[0176] In one specific embodiment, the heavy chain variable region comprises the variable regions HCDR1, HCDR2, and HCDR2 shown in SEQ ID NO:49.

[0177] HCDR3; the light chain variable region includes the variable regions LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:50.

[0178] In some embodiments, the second antigen-binding module comprises a heavy chain variable region having an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:49, and a light chain variable region comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:50. In one specific embodiment, the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:49, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50. In a more specific embodiment, the heavy chain variable region and the light chain variable region of the second antigen-binding module are shown in SEQ ID NO:49 and 50, respectively.

[0179] Table S5 shows exemplary CDR and variable region sequences of the second antigen-binding module.

[0180]

[0181] The multispecific antibody of the present invention may be without an Fc domain, and the first antigen binding module, the second antigen binding module, and the third antigen binding module are connected by a suitable connector.

[0182] The multispecific antibody of the present invention may have an Fc domain, which can prolong the half-life and provide Fc domain-related effectors and other functions.

[0183] Multispecific antibody configuration

[0184] The components of the multispecific antibody of the present invention can be fused together in various configurations. In some embodiments, the multispecific antibody further comprises (iv) an Fc domain consisting of two Fc polypeptides.

[0185] The third antigen-binding module may be fused with either the first antigen-binding module or the second antigen-binding module. In an alternative embodiment (1), the third antigen-binding module is fused with the first antigen-binding module, optionally via a peptide linker. In an alternative embodiment (2), the third antigen-binding module and the second antigen-binding module are fused with each other, optionally via a peptide linker.

[0186] In an alternative embodiment (1), the third antigen-binding module is fused to the first antigen-binding module, optionally via a peptide linker. More specifically, the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. Further, in some embodiments, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding module is ScFv and fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain. In other embodiments, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding module is Fab and fused at the C-terminus of its Fab heavy chain to the N-terminus of another Fc peptide in the Fc domain.

[0187] In one specific implementation, both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is a Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. This type of construction is schematically described in... Figure 5In embodiment A, a more specific embodiment further includes a multispecific antibody having three polypeptide chains. The first polypeptide chain contains the aforementioned first antigen-binding module, third antigen-binding module, and one of the Fc polypeptides of the Fc domain. The second polypeptide chain contains the Fab heavy chain of the aforementioned second antigen-binding module and another Fc polypeptide of the Fc domain. The third polypeptide chain is the Fab light chain of the aforementioned second antigen-binding module.

[0188] In another specific embodiment, both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is ScFv, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module. This type of construction is schematically described in... Figure 5 In embodiment B, a more specific embodiment further includes a multispecific antibody having two polypeptide chains. The first polypeptide chain contains the aforementioned first antigen-binding module, third antigen-binding module, and one Fc polypeptide of the Fc domain. The second polypeptide chain contains the aforementioned second antigen-binding module and another Fc polypeptide of the Fc domain.

[0189] Regarding the connection method of the third antigen-binding module, in the second alternative embodiment (2), the third antigen-binding module and the second antigen-binding module are fused together, optionally via a peptide linker. More specifically, the third antigen-binding module is fused at its C-terminus to the N-terminus of the second antigen-binding module. Further, in some embodiments, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding module is ScFv and is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain. In other embodiments, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding module is Fab and is fused at its C-terminus of the Fab heavy chain to the N-terminus of another Fc peptide in the Fc domain. In this embodiment, the third antigen-binding module is fused to the N-terminus of either the Fab light chain or the Fab heavy chain of the second antigen-binding module.

[0190] In one specific implementation, both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is ScFv, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the second antigen-binding module. This type of construction is schematically described in... Figure 5 In embodiment C, more specifically, the multispecific antibody has two polypeptide chains, wherein the first polypeptide chain contains the aforementioned first antigen-binding module and one of the Fc polypeptides of the Fc domain, and the second polypeptide chain contains the aforementioned second antigen-binding module, third antigen-binding module and another Fc polypeptide of the Fc domain.

[0191] In another specific embodiment, both the first and third antigen-binding modules are single variable domains, the second antigen-binding module is a Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc polypeptide in the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the Fab light chain of the second antigen-binding module (such a configuration is schematically described in...). Figure 5 (D) or the N-terminus of the Fab heavy chain of the second antigen-binding module. More specifically, in this embodiment, the multispecific antibody has three polypeptide chains, wherein the first polypeptide chain contains one of the Fc polypeptides of the first antigen-binding module and the Fc domain, the second polypeptide chain contains the Fab heavy chain of the second antigen-binding module and another Fc polypeptide of the Fc domain, and the third polypeptide chain contains the Fab light chain of the third antigen-binding module and the second antigen-binding module. Optionally, the multispecific antibody has three polypeptide chains, wherein the first polypeptide chain contains one of the Fc polypeptides of the first antigen-binding module and the Fc domain, the second polypeptide chain contains the Fab heavy chain of the second antigen-binding module, the third antigen-binding module and the other Fc polypeptide of the Fc domain, and the third polypeptide chain contains the Fab light chain of the second antigen-binding module.

[0192] When the second antigen-binding module is ScFv, the arrangement order of its heavy chain variable region (VH) and light chain variable region (VL) is arbitrary. For example, in one embodiment, the VH and VL of the second antigen-binding module are arranged in the order VL-VH from the N-terminus to the C-terminus; in another embodiment, the VH and VL of the second antigen-binding module are arranged in the order VH-VL from the N-terminus to the C-terminus. In some embodiments, the VH and VL of the second antigen-binding module are fused via a peptide linker to form ScFv.

[0193] In any of the above embodiments, the components of the multispecific antibody can be operatively linked, either directly or via various peptide linkers (e.g., peptide linkers containing one or more amino acids, typically about 1-50 amino acids), hinge fusion, as can be reasonably chosen by those skilled in the art.

[0194] The third antigen-binding module can be fused to the first antigen-binding module or the second antigen-binding module directly or via a peptide linker. In one embodiment, the third antigen-binding module is fused to the first antigen-binding module or the second antigen-binding module via a peptide linker. In some embodiments, when the second antigen-binding module has an ScFv structure, its light chain variable region and heavy chain variable region are connected via a peptide linker.

[0195] Each peptide linker can be independently adopted from any suitable one, for example, charged and / or flexible linker peptides can be used. In a specific embodiment, the peptide linker consists of 1 to 50 amino acids linked by peptide bonds, wherein said amino acids may be selected from 20 naturally occurring amino acids; in a more preferred embodiment, the 1 to 50 amino acids are selected from glycine, alanine, proline, serine, asparagine, glutamine, and lysine. Thus, exemplary peptide linkers may be polyglycine (especially (Gly)4, (Gly)5), poly(Gly-Ser), (Gly)3AsnGlySer(Gly)2, (Gly)3Cys(Gly)4, GlyProAsnGlyGly, or those disclosed in Table 4 of patent application WO2019195535, etc.

[0196] In some embodiments, the peptide linker may be a peptide linker composed of glycine and serine. In some embodiments, the number of amino acids contained in the peptide linker may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20. In some embodiments, the peptide linker is a peptide linker containing GGGGS, a peptide linker containing GGSGGSGGSGGSGG (SEQ ID NO: 2), or a peptide linker containing GGGPGKR. Preferably, the peptide linker in units of GGGGS is (GGGGS)n, where n is any number between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or any range defined by any two of the aforementioned numbers, such as 1-5, 2-5, 3-6, 2-4, 1-4, etc. In some specific embodiments, the peptide linker is a linker polypeptide comprising GGGGS, (GGGGS)2, (GGGGS)3, or (GGGGS)4. In some specific embodiments, the third antigen-binding module is fused to the first antigen-binding module or the second antigen-binding module via peptide linkers GGGGS (SEQ ID NO:1) or (GGGGS)2 or (GGGGS)3. In some embodiments, when the second antigen-binding module is an ScFv structure, its light chain variable region and heavy chain variable region are connected via peptide linkers GGGGS or (GGGGS)2 or (GGGGS)3.

[0197] When fused with Fc, fusion is typically via the hinge region. In one embodiment, the first antigen-binding module fuses with one of the Fc peptides in the Fc domain via a first hinge, and the second antigen-binding module fuses with one of the Fc peptides in the Fc domain via a second hinge. In some embodiments, the first and second hinges are capable of forming covalent bonds, such as disulfide bonds, with each other. The first and / or second hinges may contain amino acids from the hinge region of human IgG, which contains a native hinge region or a variant thereof. In some embodiments, the first and / or second hinges contain amino acids from the hinge region of human IgG1; in some embodiments, the first and / or second hinges contain amino acids from the hinge region of human IgG4. In some specific embodiments, the first hinge contains GEPKSSDKTHTCPPCP (SEQ ID NO:3), and the second hinge contains EPKSCDKTHTCPPCP (SEQ ID NO:4); in other specific embodiments, the first hinge contains EPKSCDKTHTCPPCP, and the second hinge contains GEPKSSDKTHTCPPCP. In some specific implementations, both the first hinge and the second hinge contain GEPKSSDKTHTCPPCP or EPKSCDKTHTCPPCP.

[0198] Fc domain

[0199] The Fc domain of a multispecific antibody consists of a pair of polypeptide chains containing heavy chain domains of immunoglobulin molecules. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each Fc polypeptide containing CH2 and CH3 of the constant region of the IgG heavy chain. The two Fc polypeptides of the Fc domain can stably associate with each other. In one embodiment, the multispecific antibody of the present invention comprises one Fc domain.

[0200] In one embodiment, the Fc domain of the multispecific antibody is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228, specifically an amino acid substitution at S228P, which reduces in vivo Fab arm exchange of the IgG4 antibody. In yet another specific embodiment, the Fc domain is a human Fc domain. In a more specific embodiment, the Fc domain is a human IgG1 Fc domain.

[0201] In some embodiments, the Fc domain contains modifications, such as amino acid substitutions. These modifications may include, for example, modifications that promote heterodimerization, modifications that alter effector function, or modifications that change the binding affinity to protein A.

[0202] In some implementations, the Fc includes modifications that promote heterodimerization.

[0203] The multispecific antibody of the present invention comprises a different antigen-binding module fused to one or the other of two Fc polypeptides in the Fc domain; therefore, the two Fc polypeptides are typically contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides yield several possible combinations of the two polypeptides. To improve the yield and purity of the multispecific antibody in recombinant production, it is advantageous to introduce modifications into the Fc domain of the multispecific antibody that promote the binding of the desired polypeptide. Therefore, in a specific embodiment, the Fc domain comprises amino acid substitutions that promote association between the two Fc polypeptides in the Fc domain.

[0204] The most extensive protein-protein interaction between the two Fc polypeptides of the human IgG Fc domain occurs in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is performed in the CH3 domain of the Fc domain.

[0205] In a specific implementation, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two Fc polypeptides in the Fc domain and a "mortar" modification in the other of the two Fc polypeptides in the Fc domain. Typically, this method involves introducing a protrusion ("knob") at the interface of one Fc polypeptide and a corresponding depression ("mortar") at the interface of the other Fc polypeptide, such that the protrusion can be positioned within the depression to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of one Fc polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary depression of the same or similar size as the protrusion is created at the interface of the other Fc polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0206] Therefore, in a specific implementation, in the CH3 domain of one Fc peptide of the multispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the Fc peptide that can be positioned in the CH3 domain of another Fc peptide. In the CH3 domain of the other Fc peptide, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a depression in the CH3 domain of the Fc peptide.

[0207] In some specific embodiments, one Fc polypeptide of the Fc domain comprises T366Y / W and / or S354C, and the other Fc polypeptide comprises Y407T / V, Y349C, T366S and / or L368A. In a more specific example, one Fc polypeptide of the Fc domain comprises amino acid substitutions for T366Y / W and S354C, and the other Fc polypeptide comprises amino acid substitutions for Y407T / V, Y349C, T366S, and L368A. In a more specific example, the Fc may be the Fc of human IgG1.

[0208] In some embodiments, the Fc domain includes modifications that alter effector function. In some specific embodiments, the Fc domain of the multispecific antibody is modified to reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function compared to the unmodified Fc domain. Reducing the binding affinity of the Fc domain to the Fc receptor and / or the effector function is beneficial for improving cytokine release and side effects.

[0209] In some embodiments, modifications that reduce the binding affinity of the Fc domain to the Fc receptor and / or decrease effector function are amino acid substitutions. In some embodiments, the Fc domain contains amino acid substitutions at one or more positions selected from E233, L234, L235, N297, P331, and P329. In some embodiments, the Fc domain contains amino acid substitutions at one or more positions selected from L234, L235, and P329. In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A. In one such embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc. In some embodiments, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A, P329R, or P329G. In some embodiments, the Fc domain contains an amino acid substitution at position P329 and another amino acid substitution selected from positions E233, L234, L235, N297, and P331. In a more specific embodiment, the other amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In some embodiments, the Fc domain contains amino acid substitutions at positions P329, L234, and L235. In a more specific embodiment, the Fc domain contains amino acid substitutions L234A, L235A, and P329G (“P329G LALA”). In another more specific embodiment, the Fc domain contains amino acid substitutions L234A, L235A, and P329A (“P329ALALA”). In one such embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc.

[0210] In some specific embodiments, the Fc domain is a human IgG1 Fc domain containing amino acid substitutions for L234A, L235A, and P329A / G / R.

[0211] The amino acid substitutions described above that reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function occur on both polypeptide chains of the Fc domain.

[0212] In some embodiments, the Fc domain includes a modification that reduces or eliminates the binding of the CH3 region of one Fc polypeptide to protein A (from Staphylococcus aureus). In some embodiments, this modification is an amino acid substitution. In some embodiments, the Fc domain includes amino acid substitutions H435R and / or Y436F, which occur only on one Fc polypeptide and not on the other. In one specific embodiment, the Fc domain includes amino acid substitutions H435R and / or Y436F occurring only on one of the Fc polypeptides. In such a specific embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc.

[0213] In the multispecific antibody of the present invention, the Fc domain may include one, two, or three of the following modifications: modifications that promote heterodimerization, modifications that alter effector function, and modifications that reduce or eliminate the binding of the CH3 region of one Fc polypeptide to protein A. In some embodiments, the Fc includes modifications that promote the association of two Fc polypeptides, modifications that reduce the binding affinity of the Fc domain to the Fc receptor and / or reduce effector function, and modifications that reduce or eliminate the binding of the CH3 region of one Fc polypeptide to protein A. Specific schemes for the above different modification types can be combined. For example, in one specific embodiment, the Fc domain includes the following amino acid substitutions:

[0214] i. Y407T / V, Y349C, T366S, L368A, T366Y / W, and S354C; wherein, amino acid substitutions of T366Y / W and S354C are on the same Fc polypeptide, and are not on the same Fc polypeptide as the other amino acid substitutions in (i.);

[0215] ii. L234A, L235A, and P329A; and,

[0216] iii. H435R and Y436F occur on only one of the Fc peptides.

[0217] In a more specific embodiment, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide comprises amino acid substitutions: L234A, L235A, P329A, S354C, T366W, H435R, and Y436F. In this specific embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc.

[0218] In the context of this invention, amino acid substitution is represented as: original amino acid - position - substituted amino acid, using a three-letter code (Xaa) or a single-letter code (X) to represent the amino acid residue. Therefore, for example, "H435R" means that the amino acid H at position 435 is replaced with amino acid R; the substituted amino acid can contain more than one, for example, T366Y / W means that the amino acid T at position 366 is replaced with amino acid Y or W.

[0219] In some embodiments, the multispecific antibody of the present invention is trivalent, that is, the first antigen-binding module, the second antigen-binding module, and the third antigen-binding module each provide monovalent binding to the corresponding antigen.

[0220] This invention provides an exemplary trivalent multispecific antibody.

[0221] As an example, a multispecific antibody consists of two polypeptide chains, the amino acid sequences of which are shown in SEQ ID NO:65 and SEQ ID NO:67, respectively. In some embodiments, the nucleotide sequences encoding the two polypeptide chains are shown in SEQ ID NO:66 and SEQ ID NO:68, respectively.

[0222] As an example, a multispecific antibody consists of two polypeptide chains, the amino acid sequences of which are shown in SEQ ID NO:69 and SEQ ID NO:71, respectively. In some embodiments, the nucleotide sequences encoding the two polypeptide chains are shown in SEQ ID NO:70 and SEQ ID NO:72, respectively.

[0223] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in SEQ ID NO:59, SEQ ID NO:61, and SEQ ID NO:63, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are shown in SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:64, respectively.

[0224] As an example, a multispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in SEQ ID NO:73, SEQ ID NO:61, and SEQ ID NO:75, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are shown in SEQ ID NO:74, SEQ ID NO:62, and SEQ ID NO:76, respectively.

[0225] Composition

[0226] This invention provides pharmaceutical compositions comprising multispecific antibodies and further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0227] isolated nucleic acids

[0228] This invention provides isolated nucleic acids containing polynucleotides encoding the multispecific antibodies described herein. The sequence listing provides exemplary examples of nucleic acid sequences for some multispecific antibodies.

[0229] carrier

[0230] This invention provides a vector comprising the aforementioned nucleic acid. In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector, and as a specific example, the expression vector is pcDNA3.1(+). The expression vector may optionally be any expression vector capable of expressing the multispecific antibodies described herein.

[0231] host cells

[0232] In some embodiments, the present invention provides a host cell comprising the vector of the present invention, wherein the host cell is a suitable host cell for cloning or expressing multispecific antibodies. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing multispecific antibodies. Mammalian cells are, for example, Chinese hamster ovary (CHO) cells or CHO-S cells.

[0233] Methods for preparing multispecific antibodies

[0234] In some embodiments, the present invention provides a method for preparing multispecific antibodies, the method comprising: culturing host cells to express the multispecific antibody, and isolating and purifying the multispecific antibody in the system. To generate the multispecific antibody, nucleic acid encoding the multispecific antibody is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The nucleic acid can be obtained using various methods well known in the art, such as gene splicing and chemical synthesis.

[0235] use

[0236] This invention provides the use of multispecific antibodies. In some specific embodiments, the multispecific antibodies used may be BC24, PC1, PC2, and / or PC3.

[0237] This invention provides the use of the aforementioned multispecific antibody or the aforementioned pharmaceutical composition in the preparation of medicaments for treating BCMA expression-related diseases.

[0238] The present invention provides a method for treating BCMA expression-related diseases in subjects, comprising administering a therapeutically effective amount of the multispecific antibody or the pharmaceutical composition thereof to the subjects.

[0239] In some implementations, the disease is a tumor or an autoimmune disease.

[0240] In some implementations, the disease is lymphoma, multiple myeloma, leukemia, systemic lupus erythematosus, or rheumatoid arthritis.

[0241] The present invention also provides exemplary embodiments:

[0242] Implementation Scheme 1. A multispecific antibody comprising

[0243] (i) The first antigen binding module that binds to the first antigen;

[0244] (ii) the second antigen-binding module that binds to the second antigen; and,

[0245] (iii) The third antigen-binding module that binds to the first antigen;

[0246] Wherein the first antigen is BCMA and the second antigen is an activating T cell antigen, and both the first antigen-binding module and the third antigen-binding module are single variable domains and each independently contains any one of the following:

[0247] (a) CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9;

[0248] (b) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12;

[0249] (c) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15; or

[0250] (d) CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence shown in SEQ ID NO:18.

[0251] Implementation Scheme 2. The multispecific antibody according to Implementation Scheme 1, wherein the first antigen-binding module and the third antigen-binding module each independently include any one of the following:

[0252] (b) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12; or

[0253] (c) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15.

[0254] Implementation Scheme 3. The multispecific antibody according to Implementation Scheme 2, wherein the first antigen-binding module comprises the following complementarity-determining regions: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and the third antigen-binding module comprises the following complementarity-determining regions: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15.

[0255] Implementation Scheme 4. The multispecific antibody according to Implementation Scheme 2, wherein the third antigen binding module comprises the following complementarity-determining regions: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and the first antigen binding module comprises the following complementarity-determining regions: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15.

[0256] Implementation Scheme 5. According to the multispecific antibody described in Implementation Scheme 1, the first antigen binding module and the third antigen binding module each independently contain CDR1, CDR2 and CDR3 of a single variable domain as shown in SEQ ID NO: 19, 21, 23, 25, 37, 38, 39 or 40; preferably, the first antigen binding module and the third antigen binding module each independently contain CDR1, CDR2 and CDR3 of a single variable domain as shown in SEQ ID NO: 37 or 39.

[0257] Implementation Scheme 6. The multispecific antibody according to any one of Implementation Schemes 1-5, wherein the first antigen-binding module and the third antigen-binding module each independently comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 19, 41, 42, or 25.

[0258] Implementation Scheme 7. The multispecific antibody according to any one of Implementation Schemes 1-6, wherein the first antigen binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:41.

[0259] Implementation Scheme 8. The multispecific antibody according to Implementation Scheme 7, wherein the first antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 21, 37, or 38; preferably, the first antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 37.

[0260] Implementation Scheme 9. The multispecific antibody according to any one of Implementation Schemes 1-8, wherein the third antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:42.

[0261] Implementation Scheme 10. The multispecific antibody according to Implementation Scheme 9, wherein the third antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 23, 39, or 40; preferably, the third antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 39.

[0262] Implementation Scheme 11. The multispecific antibody according to any one of Implementation Schemes 1-6, wherein the first antigen binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:42.

[0263] Implementation Scheme 12. The multispecific antibody according to Implementation Scheme 11, wherein the first antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 23, 39, or 40; preferably, the first antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 39.

[0264] Implementation Scheme 13. The multispecific antibody according to any one of Implementation Schemes 1-6 and 11-12, wherein the third antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:41.

[0265] Implementation Scheme 14. The multispecific antibody according to Implementation Scheme 13, wherein the third antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 21, 37, or 38; preferably, the third antigen-binding module comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 37.

[0266] Implementation Scheme 15. The multispecific antibody according to any one of Implementation Schemes 1-5, wherein the first antigen-binding module and the third antigen-binding module are selected from any one of the following:

[0267] (1) A first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:21 and a third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:23, 39 or 40;

[0268] (2) A first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:37 and a third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:23, 39 or 40;

[0269] (3) A first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:38 and a third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:23, 39 or 40;

[0270] (4) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:21 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:23, 39 or 40;

[0271] (5) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:37 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:23, 39 or 40;

[0272] (6) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:38 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:23, 39 or 40;

[0273] (7) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:41 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:42;

[0274] (8) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:42 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:41;

[0275] (9) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:37 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:39; or,

[0276] (10) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:39 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:37.

[0277] Implementation Scheme 16. The multispecific antibody according to any one of Implementation Schemes 1-15, wherein the single variable domain is of camel origin or humanized.

[0278] Implementation Scheme 17. The multispecific antibody according to any one of Implementation Schemes 1-16, wherein the first antigen-binding module and the third antigen-binding module are identical.

[0279] Implementation Scheme 18. A multispecific antibody according to any one of Implementation Schemes 1-16, wherein the first antigen-binding module and the third antigen-binding module are different.

[0280] Implementation Scheme 19. The multispecific antibody according to any one of Implementation Schemes 1-18, wherein the second antigen binding module is Fab, ScFv or ScFab.

[0281] Implementation Scheme 20. The multispecific antibody according to any one of Implementation Schemes 1-19, wherein the activating T cell antigen is CD3.

[0282] Implementation Scheme 21. The multispecific antibody according to any one of Implementation Schemes 1-20, wherein the second antigen-binding module comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following complementarity-determining regions: HCDR1 comprising the amino acid sequence shown in SEQ ID NO:43, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:44, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:45; the light chain variable region comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO:46, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:47, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:48.

[0283] Implementation Scheme 22. The multispecific antibody according to any one of Implementation Schemes 1-20, wherein the second antigen-binding module comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the variable region shown in SEQ ID NO:49; and the light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the variable region shown in SEQ ID NO:50.

[0284] Implementation Scheme 23. The multispecific antibody according to any one of Implementation Schemes 1-22, wherein the second antigen-binding module comprises a heavy chain variable region having an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:49, and a light chain variable region having an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:50; preferably, the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:49, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO:50.

[0285] Implementation Scheme 24. The multispecific antibody according to any one of Implementation Schemes 1-23, wherein the second antigen binding module is murine, chimeric, or humanized.

[0286] Implementation Scheme 25. The multispecific antibody according to any one of Implementation Schemes 1-24, wherein the third antigen-binding module and the first antigen-binding module are fused together, optionally fused together via a peptide linker.

[0287] Implementation Scheme 26. The multispecific antibody according to Implementation Scheme 25, wherein the third antigen-binding module is fused to the N-terminus of the first antigen-binding module at its C-terminus.

[0288] Implementation Scheme 27. The multispecific antibody according to any one of Implementation Schemes 1-24, wherein the third antigen-binding module and the second antigen-binding module are fused together, optionally via a peptide linker.

[0289] Implementation Scheme 28. The multispecific antibody according to Implementation Scheme 27, wherein the third antigen-binding module is fused to the N-terminus of the second antigen-binding module at its C-terminus.

[0290] Implementation Scheme 29. The multispecific antibody according to any one of Implementation Schemes 1-28, further comprising:

[0291] (iv) The Fc domain consisting of two Fc polypeptides.

[0292] Implementation Scheme 30. The multispecific antibody according to Implementation Scheme 29, wherein the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, and the second antigen-binding module is ScFv and the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain.

[0293] Implementation Scheme 31. The multispecific antibody according to Implementation Scheme 30, wherein the structure of the second binding module from the N-terminus to the C-terminus is VL-VH or VH-VL.

[0294] Implementation Scheme 32. The multispecific antibody according to Implementation Scheme 29, wherein the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, and the second antigen-binding module is Fab and the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain.

[0295] Implementation Scheme 33. The multispecific antibody according to Implementation Scheme 32, wherein the third antigen-binding module is fused to the N-terminus of the Fab light chain or the Fab heavy chain of the second antigen-binding module.

[0296] Implementation Scheme 34. The multispecific antibody according to Implementation Scheme 29, wherein the second antigen-binding module is ScFv, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module.

[0297] Implementation Scheme 35. The multispecific antibody according to Implementation Scheme 29, wherein the second antigen-binding module is ScFv, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the second antigen-binding module.

[0298] Implementation Scheme 36. The multispecific antibody according to Implementation Scheme 29, wherein the second antigen-binding module is Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module.

[0299] Implementation Scheme 37. The multispecific antibody according to Implementation Scheme 29, wherein the second antigen-binding module is Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of either the Fab light chain or the Fab heavy chain of the second antigen-binding module.

[0300] Implementation Scheme 38. The multispecific antibody according to any one of Implementation Schemes 29-37, wherein the Fc domain is an IgG Fc domain.

[0301] Implementation Scheme 39. The multispecific antibody according to Implementation Scheme 38, wherein the IgG Fc domain is a human IgG Fc domain, preferably the Fc domain of human IgG1 or human IgG4.

[0302] Implementation Scheme 40. The multispecific antibody according to any one of Implementation Schemes 29-39, wherein the Fc domain comprises an amino acid substitution that promotes association between two Fc polypeptides of the Fc domain.

[0303] Implementation Scheme 41. The multispecific antibody according to any one of Implementation Schemes 29-40, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions T366Y / W and S354C, and the other Fc polypeptide comprises amino acid substitutions Y407T / V, Y349C, T366S, and L368A.

[0304] Implementation Scheme 42. The multispecific antibody according to any one of Implementation Schemes 29-41, wherein the Fc domain comprises amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function.

[0305] Implementation Scheme 43. The multispecific antibody according to Implementation Scheme 42, wherein, according to EU numbering, the amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function are located at one or more positions selected from the group consisting of: L234, L235, and P329; preferably, both Fc polypeptides of the Fc domain contain amino acid substitutions L234A, L235A, and P329G, or both contain amino acid substitutions L234A, L235A, and P329A.

[0306] Implementation Scheme 44. The multispecific antibody according to any one of Implementation Schemes 29-41, wherein the Fc domain comprises amino acid substitutions that reduce or eliminate the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A.

[0307] Implementation Scheme 45. The multispecific antibody according to Implementation Scheme 44, wherein, according to the EU number, the Fc domain comprises amino acid substitutions H435R and / or Y436F occurring only in one of the Fc polypeptides.

[0308] Implementation Scheme 46. The multispecific antibody according to any one of Implementation Schemes 29-39, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide comprises amino acid substitutions: L234A, L235A, P329A, S354C, T366W, H435R, and Y436F.

[0309] Implementation Scheme 47. The multispecific antibody according to any one of Implementation Schemes 1-46 is trivalent.

[0310] Implementation Scheme 48. The multispecific antibody according to any one of Implementation Schemes 1-46, wherein the multispecific antibody comprises:

[0311] (1) Composed of two polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO:65, and the other polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO:67;

[0312] (2) Composed of two polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO:69, and the other polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO:71;

[0313] (3) Composed of three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:59; another polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:61; and a third polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:63; or,

[0314] (4) Composed of three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:73; another polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:61; and a third polypeptide chain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:75.

[0315] Implementation Scheme 49. The multispecific antibody according to Implementation Scheme 1 has the following composition:

[0316] (1) It consists of two polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO:65 and the other polypeptide chain contains the amino acid sequence shown in SEQ ID NO:67;

[0317] (2) It consists of two polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO:69 and the other polypeptide chain contains the amino acid sequence shown in SEQ ID NO:71;

[0318] (3) Composed of three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO:59, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:61, and the third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:63; or,

[0319] (4) It consists of three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO:73, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:61, and the third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:75.

[0320] Implementation Scheme 50. An isolated nucleic acid comprising a polynucleotide encoding a multispecific antibody according to any one of Implementation Schemes 1-49.

[0321] Implementation Scheme 51. A vector comprising the nucleic acid according to Implementation Scheme 50.

[0322] Implementation Scheme 52. A host cell comprising the nucleic acid according to Implementation Scheme 50, or the vector according to Implementation Scheme 51.

[0323] Implementation Scheme 53. A method for preparing a multispecific antibody according to any one of Implementation Schemes 1-49, comprising culturing the host cells described in Implementation Scheme 52 to express the multispecific antibody, and isolating and purifying the multispecific antibody in the system.

[0324] Implementation Scheme 54. A pharmaceutical composition comprising a multispecific antibody and a pharmaceutically acceptable carrier as described in any one of Implementation Schemes 1-49.

[0325] Implementation Scheme 55. A method of treating a BCMA expression-related disease in a subject, comprising administering to the subject a therapeutically effective amount of any one of Implementation Schemes 1-49 or the pharmaceutical composition of Implementation Scheme 54.

[0326] Implementation Scheme 56. The method according to Implementation Scheme 55, wherein the disease is lymphoma, multiple myeloma, leukemia, systemic lupus erythematosus, or rheumatoid arthritis.

[0327] Implementation Scheme 57. An antibody that binds to BCMA, comprising a single variable domain, said single variable domain comprising:

[0328] (1) CDR1 containing the amino acid sequence shown in SEQ ID NO:7, CDR2 containing the amino acid sequence shown in SEQ ID NO:8, and CDR3 containing the amino acid sequence shown in SEQ ID NO:9;

[0329] (2) CDR1 containing the amino acid sequence shown in SEQ ID NO:10, CDR2 containing the amino acid sequence shown in SEQ ID NO:11, and CDR3 containing the amino acid sequence shown in SEQ ID NO:12.

[0330] (3) CDR1 containing the amino acid sequence shown in SEQ ID NO:13, CDR2 containing the amino acid sequence shown in SEQ ID NO:14, and CDR3 containing the amino acid sequence shown in SEQ ID NO:15; or

[0331] (4) CDR1 containing the amino acid sequence shown in SEQ ID NO:16, CDR2 containing the amino acid sequence shown in SEQ ID NO:17, and CDR3 containing the amino acid sequence shown in SEQ ID NO:18.

[0332] Implementation Scheme 58. The BCMA-binding antibody according to Implementation Scheme 57, wherein the single variable domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences shown in SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, 40, 41, or 42.

[0333] Implementation Scheme 59. The antibody binding to BCMA according to Implementation Scheme 58, wherein the single variable domain comprises the amino acid sequence shown in SEQ ID NO: 37, 38, 39 or 40.

[0334] Implementation Scheme 60. An antibody binding to BCMA according to any one of Implementation Schemes 57-59, wherein the single variable domain is of camel origin or humanized.

[0335] Implementation Scheme 61. An antibody binding to BCMA according to any one of Implementation Schemes 57-60, wherein the antibody comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 27, 29, 31, 33, 51, 53, 55, or 57. Detailed Implementation

[0336] Example 1: Construction of anti-BCMA single-domain antibody phage display library

[0337] Recombinant human BCMA-Fc fusion protein (ACRO, product catalog No. BC7-H5254) was emulsified with complete Freund's adjuvant at a 1:1 volume ratio and administered as the initial subcutaneous multi-site immunization to Bactrian camels. Subsequently, booster immunizations were administered every two weeks by emulsifying recombinant human BCMA-Fc fusion protein with incomplete Freund's adjuvant at a 1:1 volume ratio. Serum titers of anti-human BCMA antibodies were measured after the fourth or fifth immunization. Peripheral blood was collected from Bactrian camels after multiple rounds of immunization, and peripheral blood mononuclear cells (PBMCs) were isolated. RNA was extracted from PBMCs and reverse transcribed into cDNA to amplify the V-cell antibody of the camel. H H-coded fragment.

[0338] The amplified V H The H-coding fragment was digested with PstI / NotI restriction enzymes and inserted into the phage vector pMECS (NTCC Plasmid Vector Bacterial Cell Gene Depository Center, Product Catalog No. pMECS) to construct a recombinant vector. This vector was then electroporated into *E. coli* TG1 (Lucigen, Product Catalog No. 60502-1) to obtain the original library. The V... H The H library was amplified to the logarithmic growth phase, and M13KO7 helper phage (New England Biolabs, catalog No. N0315S) was added for further amplification. The mixture was incubated overnight at 28°C with shaking at 200 rpm. The supernatant was collected by centrifugation, and 1 / 4 volume of PEG6000 / NaCl solution (20% PEG6000 (w / v), 2.5M NaCl) was added. The mixture was incubated on ice for 1-2 hours to precipitate the phage. The phage pellet was collected by centrifugation, resuspended in PBS, and stored at -80°C with 20% glycerol as a single-domain antibody phage display library.

[0339] Example 2: Screening of anti-human BCMA single-domain antibodies

[0340] Solid-phase panning was used to pan the single-domain antibody phage display library. The panned single clones were cultured and induced to express isopropyl-β-D-thiogalactoside (IPTG) to prepare the supernatant.

[0341] Selected clones were identified positively using an indirect ELISA method targeting human BCMA-His (ACRO, catalog No. BCA-H522y). Positive clones that bound only human BCMA-His and exhibited high signal values ​​were selected for preservation and sequencing. Positive clones 1A1, 1A10, 1A11, and 1B10 were obtained. Sequence analysis revealed that 1A1's V... HThe nucleotide sequence of H is SEQ ID NO:20, and the amino acid sequence is SEQ ID NO:19; V of 1A10 H The nucleotide sequence of H is SEQ ID NO:22, and the amino acid sequence is SEQ ID NO:21; V of 1A11 H The nucleotide sequence of H is SEQ ID NO:24, and the amino acid sequence is SEQ ID NO:23; V of 1B10 H The nucleotide sequence of H is SEQ ID NO:26, and the amino acid sequence is SEQ ID NO:25.

[0342] Example 3: Preparation of anti-human BCMA V H H-Fc chimeric antibody

[0343] 3.1V H Preparation of H-Fc chimeric antibodies

[0344] V of the screened positive clones H The connection between the H sequence and the human Fc region is used to construct V. H H-Fc chimeric antibody. Specifically, the obtained V H H sequence or anti-human BCMA V H The H control antibody (BM) sequence (amino acid sequence identical to SEQ ID NO:125 in CN109153731A) was inserted into the pCDNA3.1 eukaryotic expression vector containing the human IgG1 constant region (amino acid sequence SEQ ID NO:5), and Expifectamine was used. TM The CHO Transfection Kit transient expression system (Thermo Fisher Scientific Inc., Catalog No. A29129) expresses these V... HH-Fc chimeric antibodies (BM-Fc as control). After purification using a protein A affinity column, the binding of the antibodies to human TACI and BAFF-R proteins was detected using surface plasmon resonance (SPR) technology (see 3.2 for the method). The results showed that 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc specifically bound to BCMA-His protein but not to human TACI or BAFF-R proteins. Sequence analysis revealed the following full-length amino acid sequence for 1A1-Fc: SEQ ID NO:27, and the nucleotide sequence for 1A10-Fc: SEQ ID NO:29, and the nucleotide sequence for 1A11-Fc: SEQ ID NO:31, and the nucleotide sequence for 1B10-Fc: SEQ ID NO:33, and the nucleotide sequence for 1B10-Fc: SEQ ID NO:34.

[0345] 3.2 Surface plasmon resonance technique for detecting antibody binding to human TACI and BAFF-R proteins

[0346] The specificity of the anti-human BCMA VHH-Fc chimeric antibody was detected using a biomolecular interaction analysis system (GE, Biacore T200). Amino-conjugated anti-hIgG (Fc) antibody (GE, catalog No. BR-1008-39) was added to the CM5 sensor chip and diluted with running buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P-20 (w / v), pH 7.4). H H-Fc chimeric antibody was diluted to 2 μg / ml and captured for 90 s at a flow rate of 30 μl / min through the experimental channel. Human TACI or BAFF-R protein was diluted to 100 nM with running buffer and bound at a flow rate of 50 μl / min; binding signal curves were observed. No binding curves were observed for 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc, indicating they did not bind to either TACI or BAFF-R protein.

[0347] Example 4 Anti-human BCMA V H Affinity of H-Fc chimeric antibody with human and cynomolgus monkey BCMA

[0348] 4.1 Determination of the affinity of antibodies for human and cynomolgus monkey BCMA using surface plasmon resonance (SPR) technique

[0349] Anti-human BCMA V was analyzed using a biomolecular interaction analysis system (GE, Biacore T200). H Affinity assay of H-Fc chimeric antibodies. The assay revealed that 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc all had high affinity for human BCMA protein. Among them, 1A10-Fc and 1A11-Fc showed cross-reactivity with cynomolgus monkey BCMA protein, while 1A1-Fc, 1B10-Fc, and BM-Fc did not show binding to cynomolgus monkey BCMA protein.

[0350] 4.2 Flow cytometry determination of antibody-cell binding

[0351] Flow cytometry was used to detect anti-human BCMA V. H The H-Fc chimeric antibody was used to bind to target cells with different BCMA expression levels. CHO-hBCMA cells (Aikon Biomedical Technology (Suzhou) Co., Ltd., Product Catalog No. AKD001A) are a stable cell line with high BCMA expression; U266 cells (Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Product Catalog No. 3111C0001CCC000684) are a natural human myeloma cell line with moderate BCMA expression; PRMI8226 cells (Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Product Catalog No. 3111C0001CCC000083) are a natural human myeloma cell line with low BCMA expression; and HUVEC cells (ScienCell Research Laboratories, Product Catalog No. AKD001A 8000) are a human umbilical vein endothelial cell line that does not express BCMA. The antibody was serially diluted (initial concentration 126 nM, 5-fold serial dilutions, 6 concentrations) with anti-human BCMA V. H H-Fc chimeric antibody incubation for 2×10 5 Target cells were incubated on ice for 1 hour, then washed and incubated on ice for 0.5 hours with PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, catalog No. 109-116-170). After washing the cells, they were analyzed using a flow cytometer (Thermo Fisher Scientific Inc., Attune NXT).

[0352] like Figure 1A-1CAs shown in Table 1, 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc can all effectively target and bind to CHO-hBCMA cells with high BCMA expression levels and U266 cells with moderate BCMA expression levels. They also show significant binding with RPMI8226 cells with low BCMA expression levels. 1A10-Fc and 1A11-Fc bind to U266 cells more effectively than BM-Fc. Figure 1D As shown, 1A10-Fc and 1A11-Fc did not bind to HUVEC cells that do not express BCMA, but 1A1-Fc and 1B10-Fc did bind to HUVEC cells that do not express BCMA.

[0353] Table 1 Anti-human BCMA V H Affinity of H-Fc chimeric antibodies to target cells

[0354]

[0355] 4.3 Flow cytometry assay to determine the binding of antibodies to HEK293T cells transiently transfected with cynomolgus monkeys (BCMA)

[0356] The full-length BCMA gene (SEQ ID NO:6) from cynomolgus monkeys was synthesized in vitro and inserted into the pCDNA3.1 eukaryotic expression vector. Lipofectamine was used according to the manufacturer's protocol. TM The expression vector was transfected into HEK293T cells using a 3000 (Thermo Fisher Scientific Inc., Catalog No. L3000015) method. In short, HEK293T cells were transfected at a rate of 5 × 10⁶ cells / year. 5 Cells were seeded into 6-well plates. After approximately 24 hours, the culture medium containing fetal bovine serum was aspirated and the cells were washed with PBS. Transfection reagent was then added to each well at a ratio of 5 μg DNA to 7.5 μl Lipo3000. After approximately 6 hours, the medium was replaced with one containing 10% fetal bovine serum (v / v). Cells were harvested for flow cytometry analysis approximately 24 hours after transfection.

[0357] The 1A10-Fc, 1A11-Fc, and BM-Fc antibodies were serially diluted (initial concentration 126 nM, 5-fold serial dilution, 5 concentrations; sample dilution buffer served as a negative control). Each was incubated for 2 × 10⁻⁶ days. 5Transfected cynomolgus monkey BCMA and untransfected HEK293T cells were incubated on ice for 1 hour, then washed and incubated on ice for 0.5 hours with PE-labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, catalog No. 109-116-170). After washing, the cells were analyzed by flow cytometry (Thermo Fisher Scientific Inc., Attune NXT). Figure 2 In the study, HEK293T cells (HEK293T-CynoBCMA) were transiently transfected with cynomolgus monkey BCMA. Figure 2 In the study, HEK293T cells untransfected with cynomolgus monkey BCMA were used. Table 2 shows the EC50 and maximum binding MFI (Bmax) values ​​of the Fc chimeric antibody binding to HEK293T cells transiently transfected with cynomolgus monkey BCMA. The results showed that 1A10-Fc and 1A11-Fc antibodies bound to cynomolgus monkey BCMA in the cells, while the BM-Fc antibody did not bind to cynomolgus monkey BCMA.

[0358] Table 2 Anti-human BCMA V H Binding of H-Fc chimeric antibody to HEK293T cells transiently transfected from cynomolgus monkeys (BCMA)

[0359]

[0360] Example 5 Anti-human BCMA V H H-Fc chimeric antibody blocks the binding of APRIL to BCMA.

[0361] The anti-human BCMA V was serially diluted (initial concentration 252 nM, 5-fold serial dilution, 7 concentrations). HH-Fc chimeric antibodies 1A10-Fc, 1A11-Fc, and BM-Fc were mixed with 100 ng / ml biotin-conjugated recombinant BCMA-His protein (ACRO, catalog No. BCA-H522y) at a 1:1 volume ratio and incubated at room temperature for 1 hour. The mixture was then added to an ELISA plate immobilized with recombinant APRIL protein (ACRO, catalog No. APL-H5244). A control group containing only 50 ng / ml biotin-conjugated recombinant BCMA-His protein was also included. After incubation at 37°C for 1 hour, unbound biotin-conjugated BCMA-His protein was washed away. HRP-conjugated streptavidin (eBioscience, catalog No. 18-4100-51) was added, and the plate was washed 5 times before color development. The absorbance signals at 450 nm and a reference wavelength of 650 nm were measured. The blocking rate of each antibody at each concentration against the binding signal values ​​of BCMA and APRIL was calculated using the formula: Blocking rate = (Control group signal value - Sample signal value) / Control group signal value × 100%, and curve fitting was performed. The results are as follows: Figure 3 As shown in Table 3, 1A1-Fc, 1B10-Fc, 1A10-Fc and 1A11-Fc can all effectively block the binding of APRIL to BCMA.

[0362] like Figure 4A As shown, the amino acid sequences that differ between the human BCMA extracellular region (SEQ ID NO:35) and the cynomolgus monkey BCMA extracellular region (SEQ ID NO:36) are Gly6, Ala20, Ile22, Asn31, Val45, and Thr52. Clones 1A10 and 1A11 cross-react with cynomolgus monkey BCMA protein, suggesting that the possible epitopes of these two clones are located at Gln7–His19, and / or Pro23–Ser30, and / or Asn31–Ser44, and / or Thr46–Gly51. Figure 4B This is the extracellular region structure of human BCMA from the PDB database (PDB number: 2kn1). Figure 4A , 4B As shown, the extracellular region of human BCMA contains three pairs of disulfide bonds, namely Cys8-Cys21, Cys24-Cys37 and Cys28-Cys41; human BCMA mainly binds to APRIL on the β-hairpin structure (Bossen, C. et al. Semin. Immunol. 2006, 18(5):263-275). Based on this, it is speculated that the main binding sites of clones 1A10-Fc and 1A11-Fc are between Gln7 and His19, and / or between Pro23 and Ser30, and / or between Asn31 and Ser44.

[0363] Table 3 Anti-human BCMA VH H-Fc blocks the binding of APRIL to BCMA.

[0364]

[0365] Example 6 Anti-human BCMA V H Epitope Differences Among Different Clones

[0366] 6.1 ELISA (Checkerboard Method) Detection of Anti-human BCMA V H Competition among different clones

[0367] Anti-human BCMA V H The H-Fc chimeric antibody was diluted to 2 μg / ml, coated onto a high-adsorption ELISA plate, washed, and then blocked; 20 μg / ml of anti-human BCMA V was added. H H-Fc chimeric antibody and biotin-conjugated BCMA-His protein (ACRO, catalog No. BCA-H522y) were incubated at room temperature for 0.5 hours to obtain an antigen-antibody mixture. Following a checkerboard pattern, the incubated antibody-antigen mixture or biotin-conjugated BCMA-His protein alone (control group) was added sequentially at 100 μl / well to a plate and incubated at 37°C for 1 hour. Unbound biotin-conjugated BCMA-His protein was washed away, and HRP-conjugated streptavidin (eBioscience, catalog No. 18-4100-51) was added. After washing the plate 5 times, color development was performed. The absorbance signal values ​​at 450 nm and 650 nm reference wavelength were detected. The blocking rate of one antibody against the binding signal of another antibody to BCMA-Bio was calculated using the formula: Blocking rate = (Control group signal value - Sample group signal value) / Control group signal value × 100%. The results are shown in Table 4. The diagonal positions of the "\" in the chessboard (marked in gray) represent the positive control groups where antibodies compete with each other, with blocking rates all exceeding 99%. The blocking rate of 1A10-Fc against the binding of 1A11-Fc to human BCMA (32.6%) was less than 50%, and the blocking rate of 1A11-Fc against the binding of 1A10-Fc to human BCMA (21.6%) was also less than 50%, indicating that there is no significant competitive relationship between 1A10-Fc and 1A11-Fc, suggesting that 1A10-Fc and 1A11-Fc can simultaneously bind to different epitopes of the BCMA protein. Similarly, there is no significant competitive relationship between 1A10-Fc and 1A1-Fc, or between 1A10-Fc and 1B10-Fc, indicating that 1A10-Fc and 1A1-Fc can simultaneously bind to different epitopes of the BCMA protein, and 1A10-Fc and 1B10 can simultaneously bind to different epitopes of the BCMA protein. The blocking rates of 1A1-Fc, 1A11-Fc and 1B10-Fc all exceed 99%.

[0368] Table 4. ELISA (chessboard method) Epitope Competition

[0369]

[0370] 6.2 Epitope Difference Analysis Using Surface Plasmon Resonance Technology

[0371] Epitope competition analysis was performed using a biomolecular interaction analysis system (GE, Biacore T200). An amino-conjugated Anti-His Antibody (GE, Catalog No. 28995056) was added to the CM5 sensor chip. BCMA-His protein (ACRO, Catalog No. BCA-H522y) was diluted to approximately 1 μg / ml with run buffer. Capture was performed through the experimental channel at a flow rate of 30 μl / min, and the capture signal was controlled between 180 RU and 190 RU by adjusting the binding time. Anti-human BCMA V was diluted with run buffer. H H-Fc chimeric antibody V H H-Fc 1 to 10 μg / ml (saturation concentration, binding signal value remains unchanged after increasing concentration) is injected until the signal reaches the plateau. Immediately after injection, another anti-human BCMA V is injected. H H-Fc chimeric antibody V H H-Fc2. Observe the antibody binding curves and record the binding signal values ​​of the two antibodies separately. The signal value changes are shown in Table 5. The signal value of 1A10-Fc antibody reaching saturation with BCMA is 472.5 RU; at this time, when 1A11-Fc is injected, the saturation signal value is 579.0 RU, which is comparable to the saturation signal value of 653.1 RU of 1A11-Fc injected alone. The reverse is also true, and the cumulative signal values ​​of the two injection sequences are comparable, indicating that 1A10-Fc and 1A11-Fc can simultaneously bind to different epitopes on the BCMA protein.

[0372] Table 5V H Changes and analysis of SPR signal values ​​of H-Fc antibody

[0373]

[0374]

[0375] 6.3 Flow cytometry epitope difference analysis

[0376] Because the BCMA protein expressed on the cell membrane may differ from the free protein in terms of effective epitope exposure, flow cytometry was used to further confirm whether clones 1A10 and 1A11 could simultaneously bind to the BCMA protein on the cell membrane. U266 cells were used as the target cells. 20 μg / ml or 10 μg / ml of 1A10-Fc and 1A11-Fc antibodies were individually incubated for 2 × 10⁻⁶ cells / day.5 Target cells, or mix 20 μg / ml of 1A10-Fc antibody with 20 μg / ml of 1A11-Fc antibody at a 1:1 volume ratio and incubate for 2 × 10⁻⁶ days. 5 Target cells were incubated on ice for 1 hour, then washed and incubated with PE-labeled anti-human IgG Fc antibody (Jackson ImmunoResearch, catalog No. 109-116-170). After washing, the cells were analyzed using flow cytometry (Thermo Fisher Scientific Inc., Attune NXT). The results are shown in Table 6. When the concentration of the same antibody was increased by 10 μg / ml, the mean fluorescence intensity of the 1A10-Fc sample increased by 9.5% (902.0 / 823.5-1) and the mean fluorescence intensity of the 1A11-Fc sample increased by 0.6% (702.0 / 697.5-1), indicating that both antibodies were close to saturation concentration at 10 μg / ml. Based on this, adding 10 μg / ml of different antibodies, specifically 10 μg / ml of 1A10-Fc plus 10 μg / ml of 1A11-Fc, resulted in a 75.3% increase in the average fluorescence value (1443.5 / 823.5-1 = 75.3%); adding 10 μg / ml of 1A11-Fc plus 10 μg / ml of 1A10-Fc resulted in a 107.0% increase in the average fluorescence value (1443.5 / 697.5-1 = 107.0%). This indicates that the two antibodies, 1A10-Fc and 1A11-Fc, can simultaneously bind to different epitopes of the BCMA protein on the cell membrane.

[0377] Table 6. Binding of anti-human BCMAVHH chimeric antibody to U266 cells.

[0378]

[0379] Example 7 Anti-human BCMA V H Construction, expression, and purification of H-Fc chimeric monoclonal antibodies and their humanized monoclonal antibodies

[0380] For V respectively H The H-Fc chimeric antibodies 1A10-Fc and 1A11-Fc were humanized. Two humanized antibodies, 1A10-V1 and 1A10-V2, were obtained after humanizing chimeric antibody 1A11-Fc. Two humanized antibodies, 1A11-V1 and 1A11-V2, were obtained after humanizing chimeric antibody 1A11-Fc. The nucleotide and amino acid sequences of the above antibodies are shown in Table 7.

[0381] Synthetic encoding of anti-human BCMA V HThe DNA sequences of H-Fc chimeric antibodies and their humanized antibodies (SEQ ID NO: 30, 52, 54, 32, 56, 58, corresponding names are shown in Table 7) were cloned into pcDNA3.1(+) expression vectors, respectively. Each antibody was expressed using the ExpiCHO expression kit (Thermo Fisher, catalog number A29133). First, the constructed expression vectors containing the antibody DNA sequences were transfected into ExpiCHO cells (CHO-S, Thermo Scientific). The cells were then cultured in ExpiCHO expression medium at 37°C in a humidified incubator containing 8% CO2 on a tracked shaker platform rotating at 130 rpm. The culture supernatant was collected, and the protein was purified using protein A magnetic beads (Genscript, catalog number L00273). Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).

[0382] Table 7: Anti-human BCMA V H Sequence information of H-Fc chimeric monoclonal antibodies and their humanized monoclonal antibodies

[0383]

[0384]

[0385] Example 8 Anti-human BCMA V H H-Fc chimeric antibodies and their humanized antibodies bind to the human BCMA antigen.

[0386] The affinity between the test antibody and human BCMA protein was determined using a Biacore T200 (GE) analyzer. The experimental procedure is described below:

[0387] A certain amount of the test antibodies (including: 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, 1A11-V2) were captured on a CM5 chip (Cytiva, catalog 29234600) conjugated with Anti-hIgG (Cytiva, catalog 29149603). Human BCMA was then passed through the chip surface, and the reaction signal was detected in real time using Biacore software to obtain binding and dissociation curves. The buffer used in the experiment was Biacore universal buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4·12H2O, 1.8mM KH2PO4, 0.05% surfactant P-20 (w / v), pH 7.4). Anti-hIgG conjugated to the CM5 chip surface achieved a reaction value of approximately 9000 RU, capturing approximately 200 RU of the target antibodies (including 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, 1A11-V2). Then, the signal values ​​of the interaction between different concentrations of human BCMA (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM) and the target antibodies (including 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, 1A11-V2) were detected. The flow rate in the flow cell was 50 μl / min, the binding time was 240 s, the dissociation time was 1400 s, and regeneration with 3M MgCl2 (GE) was performed for 60 s, resulting in a stable baseline. The results were obtained based on the 1:1 combination pattern calculation of affinity and kinetics in the Biacore evaluation software. Anti-human BCMA V H The affinity of H-Fc chimeric antibodies and their humanized antibodies (including: 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, 1A11-V2) for human BCMA is shown in Table 8.

[0388] Table 8 Anti-human BCMA V H Binding affinity of H-Fc chimeric antibodies and humanized antibodies to human BCMA

[0389] 1A10-Fc Human-camel chimeric antibodies 0.94nM 1A10-V1 Humanized antibodies 0.337nM 1A10-V2 Humanized antibodies 1.31nM 1A11-Fc Human-camel chimeric antibodies 3.96nM 1A11-V1 Humanized antibodies 5.05nM 1A11-V2 Humanized antibodies 4.37nM

[0390] Example 9: Construction, expression, and purification of anti-BCMA / anti-CD3 multispecific antibodies

[0391] Knobs-into-holes (Ridgway, et al., 1996) Fc engineering was used to generate anti-BCMA / anti-CD3 multispecific antibodies against human IgG1. One of the polypeptide sequences in the Fc domain was designed with H435R and Y436F mutations (Jendeberg et al., 1997) to reduce the affinity of Fc for protein A, which is beneficial for removing homodimers formed during the assembly of multispecific antibodies in protein A affinity purification.

[0392] In this embodiment, four anti-BCMA / anti-CD3 multispecific antibodies of the present invention were constructed and named BC24, PC1, PC2 and PC3, respectively, wherein the conformation of BC24 is as follows. Figure 6 As shown in Figure A, the configuration of PC1 is as follows: Figure 6 As shown in Figure B, the configuration of PC2 is as follows: Figure 6 As shown in Figure C, the configuration of PC3 is as follows: Figure 6 As shown in Figure D. In the constructed multispecific antibody, the antigen-binding domain binding to BCMA is derived from anti-human BCMA V. H H-Fc chimeric antibodies and their humanized antibodies, the antigen-binding domain of CD3 was obtained by humanizing sp34 antibody (Silvana Pessano, et al., The T3 / T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits, EMBO J. 1985 Feb; 4(2): 337–344.).

[0393] The anti-BCMA / anti-CD3 multispecific antibody BC24 has three polypeptide chains. One polypeptide chain (named A10-linker-A11-Fc) contains two tandem antigen-binding domains that bind BCMA. These two antigen-binding domains are derived from the single variable domains of 1A10-Fc and 1A11-Fc, respectively. The amino acid sequence of A10-linker-A11 of this polypeptide chain is shown in SEQ ID NO:59, and the nucleotide sequence is shown in SEQ ID NO:60. The other two polypeptide chains of the anti-BCMA / anti-CD3 multispecific antibody BC24 form an antigen-binding domain in Fab form that binds CD3. These two polypeptide chains are named anti-CD3-HC-Fc (amino acid sequence shown in SEQ ID NO:61, nucleotide sequence shown in SEQ ID NO:62) and anti-CD3-LC (amino acid sequence shown in SEQ ID NO:63, nucleotide sequence shown in SEQ ID NO:64), respectively.

[0394] The anti-BCMA / anti-CD3 multispecific antibody PC1 has two polypeptide chains. One polypeptide chain (named anti-BCMA-A10-v1-linker-A11-v1-Fc) contains two tandem BCMA-binding antigen-binding domains derived from the single variable domains of humanized antibodies 1A10-V1 and 1A11-V1, respectively. The N-terminus of one polypeptide chain of Fc is fused to the BCMA-binding antigen-binding domain (derived from 1A11-V1). The amino acid sequence of this polypeptide chain anti-BCMA-A10-v1-linker-A11-v1-Fc is shown in SEQ ID NO:65, and the nucleotide sequence is shown in SEQ ID NO:65. As shown in NO:66, another polypeptide chain of the anti-BCMA / anti-CD3 multispecific antibody PC1 (named anti-CD3-scFv-Fc) contains an antigen-binding domain that binds to CD3. This antigen-binding domain is formed by the tandem formation of a CD3-binding heavy chain variable region and a light chain variable region. The N-terminus of another polypeptide of Fc is fused with the CD3-binding antigen-binding domain. The amino acid sequence of this polypeptide chain anti-CD3-scFv-Fc is shown in SEQ ID NO:67, and the nucleotide sequence is shown in SEQ ID NO:68.

[0395] The anti-BCMA / anti-CD3 multispecific antibody PC2 has two polypeptide chains. One polypeptide chain (named anti-BCMA-A10-v1-Fc) contains an antigen-binding domain that binds to BCMA. This antigen-binding domain is derived from the single variable domain of the humanized antibody 1A10-v1. The N-terminus of one polypeptide chain of Fc is fused to the antigen-binding domain that binds to BCMA. The amino acid sequence of the polypeptide chain anti-BCMA-A10-v1-Fc is shown in SEQ ID NO:69, and the nucleotide sequence is shown in SEQ ID NO:69. As shown in NO:70, another polypeptide chain of the anti-BCMA / anti-CD3 multispecific antibody PC2 (named anti-BCMA-A11-v1-linker-anti-CD3-scFv-Fc) contains a CD3-binding antigen-binding domain and a BCMA-binding antigen-binding domain in tandem. The CD3-binding antigen-binding domain is formed by the tandem heavy chain variable region and light chain variable region of CD3-binding to form the ScFv structure. The BCMA-binding antigen-binding domain is derived from the single variable domain of the humanized antibody 1A11-v1. The N-terminus of another polypeptide of Fc is fused with the CD3-binding antigen-binding domain. The amino acid sequence of this polypeptide chain anti-BCMA-A11-v1-linker-anti-CD3-scFv-Fc is shown in SEQ ID NO:71, and the nucleotide sequence is shown in SEQ ID NO:72.

[0396] The anti-BCMA / anti-CD3 multispecific antibody PC3 has three polypeptide chains. One of these chains (named anti-BCMA-A11-v1-Fc) contains a BCMA-binding antigen-binding domain derived from the single variable domain of the humanized antibody 1A11-v1. The N-terminus of one polypeptide chain of Fc is fused to the BCMA-binding antigen-binding domain. The amino acid sequence of the anti-BCMA-A11-v1-Fc polypeptide chain is shown in SEQ ID NO:73, and the nucleotide sequence is shown in SEQ ID NO:73. As shown in NO:74, the other two polypeptide chains of the anti-BCMA / anti-CD3 multispecific antibody PC3 form an antigen-binding domain in the form of Fab containing CD3. The N-terminus of the Fab light chain is fused with the antigen-binding domain that binds BCMA, which is derived from the single variable domain of the humanized antibody 1A10-v1. The N-terminus of the other polypeptide of Fc is fused with the Fab heavy chain containing the antigen-binding domain that binds CD3. These two polypeptide chains are named anti-CD3-HC-Fc (amino acid sequence as shown in SEQ ID NO:61, nucleotide sequence as shown in SEQ ID NO:62) and anti-BCMA-A10-v1-linker-anti-CD3-VL (amino acid sequence as shown in SEQ ID NO:75, nucleotide sequence as shown in SEQ ID NO:76), respectively.

[0397] In this embodiment, Celgene's anti-BCMA / anti-CD3 bispecific antibody was used as the control antibody, with the sequence source US20190263920A1. The anti-BCMA / anti-CD3 bispecific control antibody Benchmark (Celgene BM) in this embodiment contains two Fab-type antigen-binding domains that bind BCMA and one Fab-type antigen-binding domain that binds CD3. One anti-BCMA arm is in the form of an anti-BCMA-Fab-Fc structure, including the polypeptide chains anti-BCMA-HC-Fc (amino acid sequence as shown in SEQ ID NO:77, nucleotide sequence as shown in SEQ ID NO:78) and anti-BCMA-LC (amino acid sequence as shown in SEQ ID NO:79, nucleotide sequence as shown in SEQ ID NO:80), and is located in the anti-BCMA-HC-Fc (SEQ ID NO:78). The H435R and Y436F point mutations were added to NO:77 to improve the purification yield; the other anti-BCMA / anti-CD3 arm of the anti-BCMA / anti-CD3 bispecific control antibody BM has an anti-BCMA-Fab-CD3-Fab-Fc structure, including the polypeptide chains anti-BCMA–VH-anti-CD3-VL-Fc (amino acid sequence as shown in SEQ ID NO:81, nucleotide sequence as shown in SEQ ID NO:82), anti-CD3-VH-CL (amino acid sequence as shown in SEQ ID NO:83, nucleotide sequence as shown in SEQ ID NO:84), and anti-BCMA-LC (amino acid sequence as shown in SEQ ID NO:79, nucleotide sequence as shown in SEQ ID NO:80).

[0398] For each anti-BCMA / anti-CD3 antibody, the DNA sequence encoding each polypeptide chain that makes up the antibody is inserted into a pCDNA3.1(+) vector to obtain an expression vector expressing the corresponding polypeptide chain. Protein expression is driven by the CMV promoter. Polyadenylation is driven by a synthetic polyA signal sequence located at the 3' end of the CDS. In addition, each vector contains an Ori sequence for autonomous replication.

[0399] To generate these antibody molecules, each antibody combination was expressed using the ExpiCHO Expression Kit (Thermo Fisher, catalog number A29133). First, the corresponding expression vectors were transfected at specific ratios: in PC1, the transfection ratio was 1:1.5 for vector (anti-BCMA-A10-v1-linker-A11-v1-Fc) to vector (anti-CD3-scFv-Fc); in PC2, the transfection ratio was 1:1.5 for vector (anti-BCMA-A10-v1-Fc) to vector (anti-BCMA-A11-v1-linker-anti-CD3-scFv-Fc); and in PC3, the transfection ratio was 1:1.5:1.5 for vector (anti-BCMA-A11-v1-Fc) to vector (anti-CD3-HC-Fc) to vector (anti-BCMA-A10-v1-linker-anti-CD3-VL). The transfection ratio of BM was 1:2:1:1.5 for vector (anti-BCMA-HC-Fc): vector (anti-BCMA-LC): vector (anti-BCMA-VH-anti-CD3-VL-Fc): vector (anti-CD3-VH-CL); the transfection ratio of BC24 was 1:1.5:2 for vector (A10-linker-A11-Fc): vector (anti-CD3-HC-Fc): vector (anti-CD3-LC). The transfected cells were then cultured in ExpiCHO expression medium at 37°C in a humidified incubator containing 8% CO2 on a tracked shaker platform rotating at 130 rpm. The cell culture supernatant was harvested after 10-14 days of culture, and the cells were removed by centrifugation. The supernatant was purified by affinity chromatography, ion exchange chromatography and gel chromatography on an AKTApurifier 100 (GE) system.

[0400] The protein concentration of a purified protein sample was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence.

[0401] The purity of samples collected in each aliquot was determined using size exclusion chromatography (SEC). Specifically, an ACQUITY UPLC Protein BEH SEC column (WATERS) was used with a running buffer of 16 mM NaH₂PO₄, 34 mM Na₂HPO₄, 200 mM NaCl, pH 7.0, at 25°C to analyze the aggregate content of molecules. Samples with a purity greater than 95% were pooled based on the SEC results.

[0402] Complete molecular weight analysis was performed using a Waters LC-MS system (Waters, Singapore, USA, UK). A MAbPac RP 4μm 2.1×50mm (Thermo, USA) column was used; the mobile phases were A (0.1% formic acid aqueous solution) and B (0.1% formic acid acetonitrile solution), and the detection wavelength was 280 nm. 1 μg of protein was injected into the LC-MS system with a gradient of 5% B to 100% B over 5.5 minutes. The mass spectrometer was in positive ion mode with a scan range of 200-4000 m / z. Data were acquired using MassLynx 4.1 and processed using UNIFI 1.8.2.169. LC-MS analysis showed that the detected molecular weights of all anti-BCMA / anti-CD3 antibody samples were consistent with the theoretical molecular weights; among them... Figure 18 The complete molecular weight detection results of PC1 are shown. The molecular weight of the main peak of the sample is 108516 Da, which is consistent with the theoretical molecular weight of PC1. No homologous impurity peaks were observed.

[0403] Table 9: Sequence information of anti-BCMA / anti-CD3 multispecific antibodies

[0404]

[0405]

[0406]

[0407]

[0408]

[0409] Example 10: Anti-BCMA / anti-CD3 multispecific antibody binds to antigen SPR

[0410] The affinity of the test antibody for human CD3e or human BCMA protein was determined using a Biacore T200 (GE) analyzer. The experimental procedure is described below:

[0411] A certain amount of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and neogenic BM) were captured using a CM5 chip (Cytiva, catalog 29149603) conjugated with Anti-hIgG (Cytiva, catalog 29234600). Human CD3e (Acro, catalog CDE-H5223) or human BCMA was then passed through the chip surface. The reaction signal was detected in real time using Biacore software to obtain binding and dissociation curves. The buffer used in the experiment was Biacore universal buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4·12H2O, 1.8mM KH2PO4, 0.05% surfactant P-20 (w / v), pH 7.4). Anti-hIgG conjugated to the CM5 chip surface achieved a reaction value of approximately 9000 RU, capturing approximately 200 RU of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and neogenic BM). The interaction signal values ​​between different concentrations of human CD3e protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM) or human BCMA (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM) and anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and neogenic BM) were then detected. The flow rate in the flow cell was 50 μL / min, the binding time was 240 s, the dissociation time was 1400 s, and regeneration with 3M MgCl2 (GE) was performed for 60 s, resulting in a stable baseline. The results were obtained based on the affinity and kinetics 1:1 binding mode calculation in the biacore evaluation software. The affinity of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and neogenic BM) for human CD3e protein or human BCMA is shown in Table 10.

[0412] Table 10 Affinity of anti-BCMA / anti-CD3 antibodies to human CD3e or human BCMA protein

[0413] BC24 0.0627nM 0.286nM PC1 0.0396nM 1.43nM PC2 0.0185nM 1.33nM PC3 0.0347nM 1.17nM Newbase BM 0.821nM 1.60nM .

[0414] Example 11: Binding of Anti-BCMA / anti-CD3 multispecific antibody to BCMA on the surface of multiple myeloma cells

[0415] The binding of Anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) to BCMA-expressing cells (NCI-H929 cells, a cell line with high BCMA expression, sourced from Nanjing Kebai Biotechnology), MM1S cells, a cell line with moderate BCMA expression, sourced from Beina Biotechnology), and RPMI-8226 cells, a cell line with low BCMA expression, sourced from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, was analyzed by flow cytometry. Negative samples were trastuzumab.

[0416] The specific experimental procedure is as follows:

[0417] Logarithmic growth phase NCI-H929 cells (BCMA+++), MM1S cells (BCMA++), and RPMI-8226 cells (BCMA+) were used to adjust the cell density to 5 × 10⁶ cells per cubic meter using RPMI medium (Hyclon, catalog number SH30809.01) containing 2% FBS (fetal bovine serum). 6 –10×10 6 Cells / ml, 50 μl per well, were seeded into 96-well U-shaped cell culture plates (Costar, catalog number: 3799). Different concentrations of the test antibody were prepared using the above-mentioned culture medium. For cells expressing high and moderate BCMA, the highest antibody concentration was 440 nM, with 9 concentration gradients of 5-fold serial dilutions. For cells expressing low BCMA, the highest antibody concentration was 2200 nM, with 10 concentration gradients of 5-fold serial dilutions. 50 μl of each antibody concentration was added to the 96-well cell culture plates, mixed, and incubated at 4°C for 1 hour. After washing the cells with pre-chilled running buffer (MACS, catalog number 130-091-221) and discarding the supernatant, 100 μl of pre-chilled fluorescently labeled goat anti-human IgG antibody (Jackson, catalog number 109-116-170) was added to each well for resuspending, mixed, and incubated at 4°C for 30 minutes. After washing, cells were resuspended in 100 μl of pre-cooled running buffer, mixed well, and then flow-through was performed on an Attune™ NxT flow cytometer. Data were analyzed using Graphpad Prism5 software.

[0418] Figure 7-9Table 11 shows the binding ability of the Anti-BCMA / anti-CD3 multispecific antibody to NCI-H929 cells (BCMA+++), MM1S cells (BCMA++), and RPMI-8226 cells (BCMA+). The results show that in target cells with high and moderate BCMA expression, PC1, PC2, PC3, and BC24 exhibited superior maximum binding to the target antigen compared to Celgene BM. In target cells with low BCMA expression, PC3 and BC24 showed superior binding to the target antigen compared to other samples.

[0419] Table 11: EC50 values ​​and maximum binding amounts of various test samples to target cells in different cell types

[0420]

[0421] Example 12: Binding of Anti-BCMA / anti-CD3 multispecific antibody to CD3 on the surface of T cells

[0422] The binding of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) to CD3 on the surface of T cells was analyzed by flow cytometry. Trastuzumab was used as a negative control.

[0423] The specific experimental procedure is as follows:

[0424] Human PBMC cells were subjected to CD3 magnetic bead (Miltenyi, catalog number 130-097-043) CD3 assay. + T cell sorting: Frozen PBMCs were thawed, centrifuged and washed twice, and then counted. Cells were sorted at 10... 7 Add 80 μl of running buffer (MACS, catalog number 130-091-221) to each cell, and add buffer at a ratio of 10 μl / 100 μl. 7 Add 20 μl of CD3 magnetic beads to each cell, mix well, and incubate at 4°C for 15 minutes. Repeat the process at 10... 7 Wash cells with 1-2 ml of buffer, centrifuge, discard the supernatant, and resuspend the cell pellet in 500 μl of buffer. Place the LS sorting column (Miltenyi, catalog number 130-042-401) on the MidiMACSStarting Kit (LS) magnetic rack (Miltenyi, catalog number 130-091-051), rinse, add cell suspension, and wash three times. Remove the LS sorting column from the magnetic rack, place it on a clean centrifuge tube, add 5 ml of buffer, and collect the sorted CD3+. + T cells. After counting, CD3... +T cells were adjusted to a density of 5 × 10⁶ cells using RPMI medium (Hyclone, catalog number SH30809.01) containing 2% FBS (fetal bovine serum). 6 -10×10 6 Cells / ml, 50 μl per well, were seeded into 96-well U-shaped cell culture plates (Costar, catalog number: 3799). Different concentrations of the test antibody were prepared using the above-mentioned culture medium, with a maximum antibody concentration of 2200 nM, and 10 concentration gradients of 5-fold serial dilutions. 50 μl of each antibody concentration was added to each well of the 96-well cell culture plate, mixed, and incubated at 4°C for 1 hour. After washing the cells with pre-chilled running buffer (MACS, catalog number 130-091-221) and discarding the supernatant, 100 μl of pre-chilled fluorescently labeled goat anti-human IgG antibody (Jackson, catalog number 109-116-170) was added to each well for resuspending, mixed, and incubated at 4°C for 30 minutes. After washing, 100 μl of pre-chilled running buffer was added to resuspend the cells, mixed, and the cells were collected using an Attune™ NxT flow cytometer. Data analysis was performed using Graphpad Prism5 software.

[0425] Figure 10 Table 12 shows the effects of Anti-BCMA / anti-CD3 multispecific antibodies on CD3. + The results showed that all anti-BCMA / anti-CD3 multispecific antibodies could bind to CD3. + T cells.

[0426] Table 12 Antibodies to be tested against CD3 + T cell binding EC50 and maximum binding amount

[0427] Celgene BM 18.12 93499 BC24 38.34 162535 PC1 485.4 92854 PC2 ～461.6 37242 PC3 85.81 98353 negative control NA 5980

[0428] Example 13: Killing of multiple myeloma target cells NCI-H929 by Anti-BCMA / anti-CD3 multispecific antibody

[0429] The cytotoxic effects of antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) on target cells (NCI-H929 BCMA+++, source: Nanjing Kebai Biotechnology) were studied using human PBMCs (peripheral blood mononuclear cells) to provide T cells. Trastuzumab was used as a negative control.

[0430] The specific experimental procedure is as follows:

[0431] The target cells, NCI-H929 cells, were adjusted to a density of 5 × 10⁶ cells using 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 Cells / ml, 50 μl per well were seeded into 96-well cell culture plates (Eppendorf, catalog number: 0030730199). Different concentrations of the test antibody were prepared using experimental culture medium, with a maximum antibody concentration of 8 nM, and 10 concentration gradients of 5-fold serial dilutions. 50 μl of each antibody concentration was added to each well of the aforementioned 96-well cell culture plates. Human PBMCs, the effector cells, were adjusted to a cell density of 2.5 × 10⁶ cells / ml using experimental culture medium. 6 Cells / ml, 100 μl per well. The following groups were set up: drug administration group (50 μl target cells + 100 μl effector cells + 50 μl antibody), target cell group (50 μl target cells + 150 μl culture medium), effector cell group (100 μl human PBMCs + 100 μl culture medium), target cell + effector cell group (50 μl target cells + 100 μl effector cells + 50 μl culture medium), blank control group (200 μl culture medium), lysis buffer control group (200 μl culture medium + 20 μl lysis buffer), and target cell maximum release group (50 μl target cells + 150 μl culture medium + 20 μl lysis buffer). The effector-to-target ratio was 10:1, and all groups were incubated for 24 hours. 45 min before detection, 20 μl / well of lysis buffer (Promega, catalog number G182A) was added to the target cell maximum release group and the lysis buffer control group. The non-radioactive cytotoxicity assay kit (Cytotox 96 non-radioactive cytotoxicity assay, Promega, G1780) was used to detect cell lysis rate.

[0432] Pyrolysis rate (%) = (OD) 给药组 -OD 靶细胞+效应细胞组 ) / (OD 靶细胞最大释放组 -OD 靶细胞组 )×100%

[0433] Figure 11Table 13 shows the lysis rate of NCI-H929 / BCMA+++ tumor cells induced by anti-BCMA / anti-CD3 multispecific antibodies. The cell-killing effect induced by anti-BCMA / anti-CD3 antibody PC1 was superior to that of PC2 and PC3, with an EC50 of approximately 0.067 nM for PC1, and approximately 0.53 nM and 0.19 nM for PC2 and PC3, respectively. BC24 and Celgene BM showed superior cell-killing effects against NCI-H929 cells compared to PC1, PC2, and PC3, with an EC50 of approximately 0.022 nM for BC24 and approximately 0.044 nM for Celgene BM.

[0434] Table 13: EC50 values ​​and maximum lysis rates of each sample on NCI-H929 cells

[0435] PC1 0.06736 36％ PC2 0.5268 38％ PC3 0.1871 35％ BC24 0.02177 40％ Celgene BM 0.04435 41％

[0436] Example 14: Killing of multiple myeloma target cells MM1S by anti-BCMA / anti-CD3 multispecific antibody

[0437] The study investigated the cytotoxic effects of antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) on target cells (MM1S BCMA++, source: BeiNa Biotechnology) by providing T cells from human PBMCs (peripheral blood mononuclear cells). Trastuzumab was used as a negative control.

[0438] The specific experimental procedure is as follows:

[0439] MM1S target cells were adjusted to a density of 5 × 10⁶ cells using 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 Cells / ml, 50 μl per well were seeded into 96-well cell culture plates (Eppendorf, catalog number: 0030730199). Different concentrations of the test antibody were prepared using experimental culture medium, with a maximum antibody concentration of 8 nM, and 10 concentration gradients of 5-fold serial dilutions. 50 μl of each antibody concentration was added to each well of the aforementioned 96-well cell culture plates. Human PBMCs, the effector cells, were adjusted to a cell density of 2.5 × 10⁶ cells / ml using experimental culture medium. 6Cells / ml, 100 μl per well. The following groups were set up: drug administration group (50 μl target cells + 100 μl effector cells + 50 μl antibody), target cell group (50 μl target cells + 150 μl culture medium), effector cell group (100 μl human PBMCs + 100 μl culture medium), target cell + effector cell group (50 μl target cells + 100 μl effector cells + 50 μl culture medium), blank control group (200 μl culture medium), lysis buffer control group (200 μl culture medium + 20 μl lysis buffer), and target cell maximum release group (50 μl target cells + 150 μl culture medium + 20 μl lysis buffer). The effector-to-target ratio was 10:1, and all groups were incubated for 24 hours. 45 min before detection, 20 μl / well of lysis buffer (Promega, catalog number G182A) was added to the target cell maximum release group and the lysis buffer control group. The non-radioactive cytotoxicity assay kit (Cytotox 96 non-radioactive cytotoxicity assay, Promega, G1780) was used to detect cell lysis rate.

[0440] Pyrolysis rate (%) = (OD) 给药组 -OD 靶细胞+效应细胞组 ) / (OD 靶细胞最大释放组 -OD 靶细胞组 )×100%

[0441] Figure 12 Table 14 shows the lysis rate of effector cells against MM1S / BCMA++ tumor cells induced by anti-BCMA / anti-CD3 multispecific antibodies. The cell-killing effect induced by the anti-BCMA / anti-CD3 multispecific antibody PC1 was superior to that induced by PC2 and PC3, with PC1 having an EC50 of approximately 0.0084 nM, while PC2 and PC3 had EC50s of approximately 0.061 nM and 0.041 nM, respectively. PC3 exhibited a better maximum lysis rate. Celgene BM showed the weakest killing effect against MM1S, with an EC50 of approximately 0.085 nM and a maximum lysis rate of 23%.

[0442] Table 14: EC50 values ​​and maximum lysis rates of MM1S cells by each sample

[0443] PC1 0.008398 26％ PC2 0.06052 28％ PC3 0.04069 36％ BC24 0.009373 21％ Celgene BM 0.08465 23％

[0444] Example 15: Killing of multiple myeloma target cells RPMI-8226 by anti-BCMA / anti-CD3 multispecific antibody

[0445] This study investigated the cytotoxic effects of antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) on target cells (RPMI-8226BCMA+, source: Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) induced by human PBMCs (peripheral blood mononuclear cells). Trastuzumab served as the negative control.

[0446] The specific experimental procedure is as follows:

[0447] The target cells, RPMI-8226 cells, were adjusted to a density of 5 × 10⁶ cells using 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 Cells / ml, 50 μl per well were seeded into 96-well cell culture plates (Eppendorf, catalog number: 0030730199). Different concentrations of the test antibody were prepared using experimental culture medium, with a maximum antibody concentration of 8 nM, and 10 concentration gradients of 5-fold serial dilutions. 50 μl of each antibody concentration was added to each well of the aforementioned 96-well cell culture plates. Human PBMCs, the effector cells, were adjusted to a cell density of 2.5 × 10⁶ cells / ml using experimental culture medium. 6 Cells / ml, 100 μl per well. The following groups were set up: drug administration group (50 μl target cells + 100 μl effector cells + 50 μl antibody), target cell group (50 μl target cells + 150 μl culture medium), effector cell group (100 μl human PBMCs + 100 μl culture medium), target cell + effector cell group (50 μl target cells + 100 μl effector cells + 50 μl culture medium), blank control group (200 μl culture medium), lysis buffer control group (200 μl culture medium + 20 μl lysis buffer), and target cell maximum release group (50 μl target cells + 150 μl culture medium + 20 μl lysis buffer). The effector-to-target ratio was 10:1, and all groups were incubated for 24 hours. 45 min before detection, 20 μl / well of lysis buffer (Promega, catalog number G182A) was added to the target cell maximum release group and the lysis buffer control group. The non-radioactive cytotoxicity assay kit (Cytotox 96 non-radioactive cytotoxicity assay, Promega, G1780) was used to detect cell lysis rate.

[0448] Pyrolysis rate (%) = (OD) 给药组 -OD 靶细胞+效应细胞组 ) / (OD 靶细胞最大释放组 -OD 靶细胞组 )×100%

[0449] Figure 13Table 15 shows the lysis rates of RPMI-8226 / BCMA+ tumor cells induced by anti-BCMA / anti-CD3 multispecific antibodies. The maximum lysis rate of RPMI-8226 by anti-BCMA / anti-CD3 multispecific antibody PC1 was comparable to that of BC24, but with a lower EC50. PC2 and PC3 showed comparable killing effects on RPMI-8226, with EC50s of 0.0185 nM and 0.0142 nM, respectively, and a maximum lysis rate of 39% for both. Celgene BM showed the weakest killing effect on RPMI-8226, with an EC50 of 0.27 nM and a maximum lysis rate of 29%.

[0450] Table 15: EC50 values ​​and maximum lysis rates of each sample on RPMI-8226 cells

[0451] PC1 0.0070 29％ PC2 0.0185 39％ PC3 0.01419 39％ BC24 0.01129 29％ Celgene BM 0.2712 29％

[0452] Example 16: T cell activation induced by anti-BCMA / anti-CD3 multispecific antibody

[0453] CD69 and CD25 are markers of T cell activation. This example involves the detection of T cell activation in the presence of myeloma target cells using multispecific antibodies (including BC24, PC1, PC2, PC3, and Celgene BM). The negative control (NC) is trastuzumab.

[0454] The specific experimental procedure is as follows:

[0455] The target cells, NCI-H929 cells and MM1S cells, were adjusted to a cell density of 5 × 10⁶ using 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 Cells / ml, 50 μl per well were seeded into 96-well cell culture plates (Eppendorf, catalog number: 0030730199). Different concentrations of the test antibody were prepared using experimental culture medium, with a maximum antibody concentration of 8 nM, and 10 concentration gradients of 5-fold serial dilutions. 50 μl of each antibody concentration was added to each well of the aforementioned 96-well cell culture plates. Human PBMCs, the effector cells, were adjusted to a cell density of 2.5 × 10⁶ cells / ml using experimental culture medium. 6Cells / ml, 100μl per well. The following groups were set up: drug administration group (50μl target cells + 100μl effector cells + 50μl antibody), no drug administration group (50μl culture medium + 100μl effector cells + 50μl antibody), target cell group (50μl target cells + 150μl culture medium), effector cell group (100μl human PBMCs + 100μl culture medium), target cell + effector cell group (50μl target cells + 100μl effector cells + 50μl culture medium), blank control group (200μl culture medium), and negative control antibody NC was trastuzumab. The effector-to-target ratio was 10:1. All groups were incubated for 24 hours. Centrifuge to remove supernatant, add pre-chilled running buffer (MACS, catalog number 130-091-221) and centrifuge to wash cells. After removing supernatant, add 100 μl of running buffer to each well and mix the cells. Add 2.5 μl each of CD4-BV421 (BD, catalog number 564713), CD8-PE-Cy7 (BD, catalog number 557746), CD25-APC (BD, catalog number 555434), and CD69-FITC (BD, catalog number 555530) to each well and incubate on ice for 30 minutes. Wash cells with pre-chilled running buffer, resuspend cells in 75 μl of running buffer per well, mix well, and then place in a flow cytometer for analysis.

[0456] Figure 14-15 The results show the activation levels of T cells in the presence and absence of NCI-H929 or MM1S target cells at an Anti-BCMA / anti-CD3 antibody concentration of 8 nM. The vertical axis represents the percentage of CD8 or CD4 cells positive for activation markers CD25 or CD69, and the horizontal axis represents different groups. Specifically, CD4+CD25+ / CD4+ indicates the percentage of CD4 cells positive for CD25 in a given group; CD4+CD69+ / CD4+ indicates the percentage of CD4 cells positive for CD69 in a given group; CD8+CD25+ / CD8+ indicates the percentage of CD8 cells positive for CD25 in a given group; and CD8+CD69+ / CD8+ indicates the percentage of CD8 cells positive for CD69 in a given group. The results indicate that T cells are essentially not activated in the absence of target cells, while antibodies BC24, PC1, PC2, and PC3 can activate T cells in their presence.

[0457] Example 17 Cytokine release induced by Anti-BCMA / anti-CD3 multispecific antibody

[0458] By detecting the levels of IL-2, IL-6, TNF-α, and IFN-γ, this study investigated the cytokine release induced by multispecific antibodies in the presence of target cells. The safety of the multispecific antibodies was assessed based on the amount of cytokine released.

[0459] The specific experimental procedure is as follows:

[0460] The target cells, NCI-H929 cells, were adjusted to a density of 5 × 10⁶ cells using 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 Cells / ml, 50 μl per well were seeded into 96-well cell culture plates (Eppendorf, catalog number: 0030730199). Different concentrations of the test antibody were prepared using experimental culture medium, with a maximum antibody concentration of 8 nM, and 10 concentration gradients of 5-fold serial dilutions. 50 μl of each antibody concentration was added to each well of the aforementioned 96-well cell culture plates. Human PBMCs, the effector cells, were adjusted to a cell density of 2.5 × 10⁶ cells / ml using experimental culture medium. 6 Cells / ml, 100μl per well. The following groups were set up: standard group, drug group (50μl target cells + 100μl effector cells + 50μl antibody), target cell group (50μl target cells + 150μl culture medium), effector cell group (100μl human PBMCs + 100μl culture medium), target cell + effector cell group (50μl target cells + 100μl effector cells + 50μl culture medium), effector cell + antibody group (100μl effector cells + 50μl antibody + 50μl culture medium), and blank control group (200μl culture medium). The effector-to-target ratio was 10:1, and the cells were incubated for 24 hours. Centrifuge and collect the supernatant. Use the human-IL2 kit (Cisbio, catalog number 62HIL02PEG), human-IL6 kit (Cisbio, catalog number 62HIL06PEG), human-TNF-α kit (Cisbio, catalog number 62HTNFAPEG), and human-IFN-γ kit (Cisbio, catalog number 62HIFNGPEG) to detect the samples according to the kit instructions. Use a microplate reader (Molecular Devices, model Paradigm) for detection and analysis.

[0461] Calculate the Ratio values ​​for all samples. Using the Ratio(Net) of the standard as the Y-axis and Log (concentration value of the standard) as the X-axis, perform four-parameter fitting using Graph Pad Prism. Substitute the Ratio(Net) values ​​of the standard and the test sample respectively to obtain the corresponding log (concentration measurement value), and calculate the concentration measurement value of each factor in the test sample.

[0462] Figure 16Table 16 shows the release levels of IL-2, IL-6, TNF-α, and IFN-γ factors after co-incubating different concentrations of Anti-BCMA / anti-CD3 antibody with NCI-H929 cells and effector cells for 24 hours. The IL-2 release of each antibody tested was low. The BC24, PC1, PC2, and PC3 multispecific antibodies of this invention exhibited lower IL-6, TNF-α, and IFN-γ factor release levels compared to Celgene BM, demonstrating better safety.

[0463] Table 16 Maximum release of cytokines from each antibody to be tested

[0464] PC1 112.06 964.63 302.56 5463.63 PC2 82.49 1 42.69 2841.61 PC3 97.51 3045.68 371.69 14391.37 BC24 88.07 260.3 200.25 4753.13 Celgene BM 117.37 3040.77 464.18 21552.72

[0465] Example 18: Growth inhibition of NCI-H929 multiple myeloma target cells by anti-BCMA / anti-CD3 bispecific antibody in a mouse tumor model.

[0466] The antitumor activity of the anti-BCMA / anti-CD3 multispecific antibody was detected using a human myeloma NCI-H929 cell mouse model. NCI-H929 cells were 7.5 × 10⁻⁶. 6 / each, hPBMC human peripheral lymphocytes 0.5×10 6 Each mouse, after mixing the two, was inoculated under aseptic conditions into the right axilla of 8-10 week old female B-NDG mice (source: Biocytogen, certificate number: 320726200100179034). Subcutaneous xenografts were implanted until the tumor volume reached 100-300 mm². 3 Animals were randomly divided into two groups: i. Model group (blank control group, physiological saline); ii. PC1 group (PC1 test antibody). Mice were grouped on day 0 and administered the drug twice a week for one week at a dose of 1.5 mpk and a volume of 10 ml / kg. Body weight and tumor diameter were measured every three days, and animal performance was observed daily until day 17. Tumor volume was measured on days 0, 4, 8, 11, 14, and 17 during the experiment. Tumor volume and tumor inhibition rate were calculated using the following formula:

[0467] Tumor volume (TV) = (length × width) 2 ) / 2;

[0468] Relative tumor volume (RTV) = TV t / TV0, where TV0 is the tumor volume when administered via a separate administration loop, and TV t The tumor volume at each measurement;

[0469] Relative tumor proliferation rate T / C (%) = (T RTV / C RTV )*100%, where TRTV For the treatment group RTV, C RTV This serves as a blank control group for RTV.

[0470] Tumor inhibition rate (TGI) = (1-T / C)*100%.

[0471] The experimental results are shown in Figure 17 Compared with the model group, the PC1 antibody drug group significantly inhibited the growth of subcutaneous xenografts of human multiple myeloma NCI-H929. The tumor inhibition rates of PC1 antibody on days 8, 11, 14, and 17 were 84.4%, 90.9%, 94.2%, and 95.5%, respectively.

[0472] Example 19 Pharmacokinetics of a single intravenous injection of an anti-BCMA / anti-CD3 multispecific antibody in cynomolgus monkeys

[0473] Cynomolgus macaques (numbered 1M01-1M03, 2M01-2M03, 3 males per dose group, weighing 2-3 kg) were administered a single intravenous infusion of PC1 injection. The PC1 multispecific antibody was administered at a dose of 1 or 9 mg / kg, with an intravenous infusion time of 30 minutes. Blood samples were collected before and after administration. Serum was separated within 1 hour by centrifugation at 4000 rpm for approximately 10 minutes (2-8℃). Drug concentration in the blood samples at each time point was detected by ELISA. Sampling times: 0 h (before administration), 0.5 h, 4 h, 24 h, 48 h, 96 h, 168 h, 240 h, and 336 h after administration.

[0474] The parameters are as follows:

[0475] C max Maximum observed drug concentration

[0476] AUC: Area under the plasma concentration-time curve

[0477] T 1 / 2 :half life

[0478] Cl: Clearance

[0479] MRT: Drug Retention Time

[0480] Experimental results showed that when Anti-BCMA / anti-CD3 multispecific antibodies were administered via a single intravenous infusion at concentrations of 1.9 mg / kg, the terminal elimination half-life (t) was [not specified]. 1 / 2 105 and 112 h respectively, C max The values ​​were 19 and 200 μg / mL, respectively, with AUC last The concentrations were 700 and 7200 h*μg / mL, respectively, and the serum exposure of this product increased in a basically proportional manner with the dose.

[0481] Table 17 Main mean pharmacokinetic parameters after a single intravenous injection of 1.9 mg / kg PC1 solution into cynomolgus monkeys.

[0482] sequence list <110> Chia Tai Tianqing Pharmaceutical Group Co., Ltd. <120> Multispecific antibodies targeting BCMA <150> 202110405638.3 <151> 2021-04-15 <160> 84 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide linkers <400> 1 Gly Gly Gly Gly Ser 1 5 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide linkers <400> 2 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Hinge area <400> 3 Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Hinge area <400> 4 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 5 <211> 232 <212> PRT <213> Homo sapiens <220> <223> Human IgG1 constant region <400> 5 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 6 <211> 183 <212> PRT <213> *Macaca fascicularis* <220> <223> Full-length cynomolgus BCMA <400> 6 Met Leu Gln Met Ala Arg Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Asp Cys Lys Pro Cys Gln Leu Arg Cys Ser Ser Thr Pro 20 25 30 Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Met Thr Asn Ser Val 35 40 45 Lys Gly Met Asn Ala Ile Leu Trp Thr Cys Leu Gly Leu Ser Leu Ile 50 55 60 Ile Ser Leu Ala Val Phe Val Leu Thr Phe Leu Leu Arg Lys Met Ser 65 70 75 80 Ser Glu Pro Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu Leu 85 90 95 Gly Met Ala Asn Ile Asp Leu Glu Lys Gly Arg Thr Gly Asp Glu Ile 100 105 110 Val Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys Glu 115 120 125 Asp Cys Ile Lys Asn Lys Pro Lys Val Asp Ser Asp His Cys Phe Pro 130 135 140 Leu Pro Ala Met Glu Glu Gly Ala Thr Ile Leu Val Thr Thr Lys Thr 145 150 155 160 Asn Asp Tyr Cys Asn Ser Leu Ser Ala Ala Leu Ser Val Thr Glu Ile 165 170 175 Glu Lys Ser Ile Ser Ala Arg 180 <210> 7 <211> 5 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A1 Single Variable Structural Domain CDR1 <400> 7 Gly Trp Asn Lys His 1 5 <210> 8 <211> 16 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A1 Single Variable Structural Domain CDR2 <400> 8 Ser Ile Phe Arg Asp Gly Lys Thr Ala Tyr Thr Asp Ser Val Lys Gly 1 5 10 15 <210> 9 <211> 10 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A1 Single Variable Structural Domain CDR3 <400> 9 Asp Leu Pro Gly Ser Gly Leu Pro Ala Phe 1 5 10 <210> 10 <211> 5 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A10 Single Variable Structural Domain CDR1 <400> 10 Val Ala Cys Met Ala 1 5 <210> 11 <211> 17 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A10 Single Variable Structural Domain CDR2 <400> 11 Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val Lys 1 5 10 15 Gly <210> 12 <211> 14 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A10 Single Variable Structural Domain CDR3 <400> 12 Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 1 5 10 <210> 13 <211> 5 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A11 Single Variable Structural Domain CDR1 <400> 13 Ser Ala Cys Met Gly 1 5 <210> 14 <211> 17 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A11 Single Variable Structural Domain CDR2 <400> 14 Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 15 <211> 17 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A11 Single Variable Structural Domain CDR3 <400> 15 Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp Tyr Asn 1 5 10 15 Tyr <210> 16 <211> 5 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1B10 Single Variable Structural Domain CDR1 <400> 16 Gly Trp Asn Lys His 1 5 <210> 17 <211> 16 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1B10 Single Variable Structural Domain CDR2 <400> 17 Ser Ile Phe Arg Asp Gly Lys Thr Ala Tyr Thr Asp Ser Val Lys Gly 1 5 10 15 <210> 18 <211> 10 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1B10 Single Variable Structural Domain CDR3 <400> 18 Asp Leu Pro Gly Ser Gly Leu Pro Glu Phe 1 5 10 <210> 19 <211> 118 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A1 Single Variable Structure Domain <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Phe Arg Gly Trp 20 25 30 Asn Lys His Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ser Ser Ile Phe Arg Asp Gly Lys Thr Ala Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Asp Ala Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Lys 85 90 95 Tyr Asp Leu Pro Gly Ser Gly Leu Pro Ala Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 20 <211> 354 <212> DNA <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A1 single variable domain <400> 20 caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ccggagggtc tctgagactc 60 tcctgtacag cctctggata caccttcagg ggctggaaca agcactggta ccgccaggct 120 ccagggaagg agcgcgagtt ggtctcgagc atctttcgtg atgggaagac agcctataca 180 gactccgtga agggccgatt caccatctct caagacaacg ccgacgcaac ggtgtatctg 240 caaatgaaca gcctgaaacc tgaggacacg gccatgtatt actgtaaata cgatctgcct 300 ggatctggtc ttcctgcatt ctggggccag gggaccctgg tcactgtctc atca 354 <210> 21 <211> 123 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1A10 single variable domain <400> 21 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 369 <212> DNA <213> *Camelus bactrianus* <220> <223> 1A10 single variable domain <400> 22 caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc gctgagactc 60 tcctgtgccg cctccggata cagcagtaat gtggcatgca tggcctggta ccgccaggct 120 ccagggaagg agcgcgagtg ggtcgcaact attgttgctg atttcggtac cacaaactat 180 gccgcctccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtgtat 240 ctgcaaatga acagcctgaa acctgaggac tctgccatgt actactgtgc ggcaacccag 300 agggggggta ttgactggtg tgatgaaatt aattactggg gccaggggac cctggtcact 360 gtctcatca 369 <210> 23 <211> 126 <212> PRT <213> *Camelus bactrianus* <220> <223> 1A11 single variable domain <400> 23 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 24 <211> 378 <212> DNA <213> Camelus bactrianus <220> <223> 1A11 single variable domain <400> 24 caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtgcag cctctggagt cacttttaat agcgcatgta tgggttggtt ccgccaggct 120 ccagggaagg agcgcgaggg ggtcgcacgt attgaaactg gttatggtgg cactgtctat 180 gccgactccg tgaagggacg attcaccatc tcccgagaca acgccaagaa cacggtgtat 240 ctgcaaatga acagcctaaa acctgaggac actgccatgt actactgtgc ggctaagaga 300 tcctggtgta ctcctacgtg gtggcacgaa cttgactata actactgggg acaggggacc 360 caggtcactg tctcatca 378 <210> 25 <211> 118 <212> PRT <213> Bactrian camel (Camelus bactrianus) <220> <223> 1B10 single variable domain <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Phe Arg Gly Trp 20 25 30 Asn Lys His Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ser Ser Ile Phe Arg Asp Gly Lys Thr Ala Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Ser Ala Asp Ala Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Lys 85 90 95 Tyr Asp Leu Pro Gly Ser Gly Leu Pro Glu Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 26 <211> 354 <212> DNA <213> Bactrian camel (Camelus bactrianus) <220> <223> 1B10 single variable domain <400> 26 gaggtgcagc tggtggagtc tgggggaggc tcggtgcagg ccggagggtc tctgagactc 60 tcctgtacag cctctggata caccttcagg ggctggaaca agcactggta ccgccaggct 120 ccagggaagg agcgcgagtt ggtctcgagt atctttcgtg atgggaagac agcctataca 180 gactccgtga agggccgatt caccatctct caagacagcg ccgacgcaac ggtgtatctg 240 caaatgaaca gcctgaaacc tgaggacacg gccatgtatt actgtaaata cgatctgcct 300 ggatctggtc ttcctgagtt ctggggccag gggaccctgg tcactgtctc atca 354 <210> 27 <211> 350 <212> PRT <213> Artificial Sequence <220> <223> 1A1-Fc <400> 27 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Phe Arg Gly Trp 20 25 30 Asn Lys His Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ser Ser Ile Phe Arg Asp Gly Lys Thr Ala Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Asp Ala Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Lys 85 90 95 Tyr Asp Leu Pro Gly Ser Gly Leu Pro Ala Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr 115 120 125 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 130 135 140 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 145 150 155 160 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 165 170 175 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 195 200 205 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 225 230 235 240 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 245 250 255 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 260 265 270 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 275 280 285 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 305 310 315 320 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 325 330 335 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 28 <211> 1050 <212> DNA <213> Artificial Sequence <220> <223> 1A1-Fc <400> 28 caagtgcaac tggtggagtc tggaggcggt tccgtgcagg ccggcggctc cctgagactg 60 tcctgcaccg cttccggcta caccttcaga ggctggaaca agcactggta tcggcaggct 120 cctggcaaag aaagagagct ggtgtcctcc atcttccggg acggcaagac cgcctacacc 180 gactccgtga agggcagatt caccatctct caggacaacg ccgacgccac agtgtacctg 240 cagatgaact ctctgaagcc cgaggacacc gccatgtact actgcaagta cgacctgcct 300 ggatctggcc tgcccgcctt ctggggccaa ggcaccctgg taaccgtgtc gtccgagccc 360 aagtcttgcg acaagaccca cacctgtcct ccttgtcctg ctccggaact gctgggcggc 420 ccttccgtgt ttctgtttcc tccaaagcct aaggacacac tgatgatcag ccggacccct 480 gaggtgacct gcgtggtcgt ggatgtctct cacgaggatc ctgaggtgaa gttcaactgg 540 tacgtggatg gagtggaagt gcataacgct aaaaccaagc ctagagaaga gcagtacaac 600 tccacctaca gagtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 660 gagtacaagt gcaaggtgtc caacaaggct ctgcctgccc ctatcgagaa gaccatctcc 720 aaggccaagg gccagcctcg ggagcctcaa gtgtacaccc tgcctccttc tcgcgacgag 780 ctgaccaaga accaggtgtc tctgacctgc ctggtgaaag gcttctaccc ctccgacatc 840 gccgtggaat gggagtccaa tggccagccc gagaacaact acaagaccac cccacctgtg 900 ctggactctg atggctcctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 960 cagcagggca acgtgttctc ctgttctgtg atgcacgagg ccctgcacaa ccactacaca 1020 cagaagtccc tgagcctgtc tcctggcaag 1050 <210> 29 <211> 355 <212> PRT <213> Artificial Sequence <220> <223> 1A10-Fc <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys 115 120 125 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 130 135 140 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 145 150 155 160 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 165 170 175 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 180 185 190 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 195 200 205 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 210 215 220 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 225 230 235 240 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 245 250 255 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 260 265 270 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 275 280 285 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 290 295 300 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 305 310 315 320 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 325 330 335 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 340 345 350 Pro Gly Lys 355 <210> 30 <211> 1065 <212> DNA <213> Artificial Sequence <220> <223> 1A10-Fc <400> 30 caggtgcaac tggtggagtc tggcggcgga tctgtgcaag ctggcggatc cctgagactg 60 tcttgtgccg cttccggcta ctcctctaac gtggcctgta tggcctggta cagacaggcc 120 cctggcaaag aaagagagtg ggtcgccacc atcgtggccg atttcggcac caccaattac 180 gccgcttccg tgaagggcag attcaccatc tctcaggaca acgccaagaa caccgtgtac 240 ctgcagatga actccctgaa gcctgaggac tccgccatgt actactgcgc cgctacacag 300 cgcggaggca ttgattggtg cgacgagatt aactactggg gccaaggcac cctggttacc 360 gtgtcctctg agcccaagtc ctgcgacaag acccacacct gtccaccttg tcctgcccca 420 gaactgctgg gcggtcccag cgtgttcctg ttccccccca agcctaagga cacactgatg 480 atctctagaa cccctgaagt gacctgcgtg gtggtcgatg tatctcacga ggatcctgag 540 gtcaagttca actggtacgt ggacggagtg gaagtgcata atgctaagac caagcctaga 600 gaggaacagt acaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat 660 tggctgaacg gcaaggagta caagtgcaag gtgtccaaca aggctctgcc tgctcctatc 720 gagaagacca tcagcaaggc caagggccag cctcggggagc ctcaagtgta caccctgcct 780 ccttctcggg acgagctgac caagaaccag gtgtctctta catgcctggt gaaaggcttc 840 tacccttccg acatcgccgt ggaatgggag tccaatggcc agcccgagaa caactacaag 900 accacacctc ctgtgctgga ctctgacggc tccttcttc tgtactccaa gctgacagtg 960 gataagtcca gatggcagca gggcaacgtg ttctcctgct ccgtgatgca cgaggccctg 1020 cacaaccact acacccagaa gtctctgagc ctgtcccctg gaaaa 1065 <210> 31 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> 1A11-Fc <400> 31 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro 115 120 125 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 130 135 140 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 145 150 155 160 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 165 170 175 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 180 185 190 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 195 200 205 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 210 215 220 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 225 230 235 240 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 245 250 255 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 260 265 270 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 275 280 285 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 290 295 300 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 305 310 315 320 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 325 330 335 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 340 345 350 Ser Leu Ser Pro Gly Lys 355 <210> 32 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> 1A11-Fc <400> 32 caggtgcagt tggttgaatc tggcggcgga tctgtgcagg ctggcggatc tctgagactg 60 tcctgtgccg cttccggcgt caccttcaac tccgcttgca tgggatggtt cagacaggcc 120 cctggcaaag agagagaggg cgtcgctagg atcgagaccg gctatggcgg aaccgtctac 180 gccgactccg tcaagggcag gttcaccatc agcagagaca acgccaagaa caccgtgtac 240 ctgcagatga actccctgaa gcccgaggac accgccatgt actactgtgc tgccaagaga 300 agctggtgca cccctacctg gtggcacgag ctggactaca actactgggg ccagggcaca 360 caggtcaccg tgtcctctga gcctaagtcc tgcgacaaga cccacacctg tcctccatgt 420 cctgctccag aactgctcgg cggaccttcc gtgttcctgt ttcctccaaa gcctaaggat 480 accctgatga tctctcggac ccctgaagtg acctgcgtgg tggtggatgt gtctcacgag 540 gatcccgaag tgaagttcaa ttggtacgtg gacggcgtgg aagtgcacaa tgctaagacc 600 aagcctagag aggaacagta caactccacc tatagagtgg tgtccgtgct gaccgtgctg 660 caccaggatt ggctgaacgg caaagagtac aagtgcaagg tgtccaacaa ggccctgcct 720 gctcctatcg aaaagaccat ctccaaggcc aagggccagc ctagggaacc ccaggtttac 780 accttgcctc catctcggga cgagctgacc aagaaccagg tgtccctgac ctgtctggtc 840 aagggcttct acccctccga tatcgccgtg gaatgggagt ctaatggcca gccagagaac 900 aactacaaga caacccctcc tgtgctggac tccgacggct cattcttcct gtactccaag 960 ctgacagtgg acaagtccag atggcagcag ggcaacgtgt tctcctgctc cgtgatgcac 1020 gaggccctgc acaatcacta cacccagaag tccctgtctc tgagccccgg caaa 1074 <210> 33 <211> 350 <212> PRT <213> Artificial Sequence <220> <223> 1B10-Fc <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Phe Arg Gly Trp 20 25 30 Asn Lys His Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ser Ser Ile Phe Arg Asp Gly Lys Thr Ala Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp Ser Ala Asp Ala Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Lys 85 90 95 Tyr Asp Leu Pro Gly Ser Gly Leu Pro Glu Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr 115 120 125 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 130 135 140 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 145 150 155 160 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 165 170 175 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 195 200 205 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 225 230 235 240 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 245 250 255 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 260 265 270 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 275 280 285 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 305 310 315 320 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 325 330 335 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 34 <211> 1050 <212> DNA <213> Artificial Sequence <220> <223> 1B10-Fc <400> 34 gaggtgcaac tggtggagtc tggaggcggt tccgtgcagg ccggcggctc cctgagactg 60 tcctgcaccg cttccggcta caccttcaga ggctggaaca agcactggta tcggcaggct 120 cctggcaaag aaagagagct ggtgtcctcc atcttccggg acggcaagac cgcctacacc 180 gactccgtga agggcagatt caccatctct caggactctg ccgacgccac agtgtacctg 240 cagatgaact ctctgaagcc cgaggacacc gccatgtact actgcaagta cgacctgcct 300 ggatctggcc tgcccgagtt ctggggccaa ggcaccctgg taaccgtgtc gtccgagccc 360 aagtcttgcg acaagaccca cacctgtcct ccttgtcctg ctccggaact gctgggcggc 420 ccttccgtgt ttctgtttcc tccaaagcct aaggacacac tgatgatcag ccggacccct 480 gaggtgacct gcgtggtcgt ggatgtctct cacgaggatc ctgaggtgaa gttcaactgg 540 tacgtggatg gagtggaagt gcataacgct aaaaccaagc ctagagaaga gcagtacaac 600 tccacctaca gagtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 660 gagtacaagt gcaaggtgtc caacaaggct ctgcctgccc ctatcgagaa gaccatctcc 720 aaggccaagg gccagcctcg ggagcctcaa gtgtacaccc tgcctccttc tcgcgacgag 780 ctgaccaaga accaggtgtc tctgacctgc ctggtgaaag gcttctaccc ctccgacatc 840 gccgtggaat gggagtccaa tggccagccc gagaacaact acaagaccac cccacctgtg 900 ctggactctg atggctcctt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 960 cagcagggca acgtgttctc ctgttctgtg atgcacgagg ccctgcacaa ccactacaca 1020 cagaagtccc tgagcctgtc tcctggcaag 1050 <210> 35 <211> 54 <212> PRT <213> Homo sapiens <220> <223> Human BCMA extracellular region <400> 35 Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30 Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45 Val Lys Gly Thr Asn Ala 50 <210> 36 <211> 53 <212> PRT <213> Macaca fascicularis <220> <223> Cynomolgus macaque BCMA extracellular region <400> 36 Met Leu Gln Met Ala Arg Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser 1 5 10 15 Leu Leu His Asp Cys Lys Pro Cys Gln Leu Arg Cys Ser Ser Thr Pro 20 25 30 Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Met Thr Asn Ser Val 35 40 45 Lys Gly Met Asn Ala 50 <210> 37 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 1A10-V1 single variable domain <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 1A10-V2 single variable domain <400> 38 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ser Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> 1A11-V1 single variable domain <400> 39 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ser Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 40 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> 1A11-V2 single variable domain <400> 40 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 41 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (11) <223> Xaa is S or L <220> <221> UNSURE <222> (14) <223> Xaa is P or A <220> <221> UNSURE <222> (44) <223> Xaa is G or E <220> <221> UNSURE <222> (45) <223> Xaa is R or L <220> <221> UNSURE <222> (49) <223> Xaa is A or S <220> <221> UNSURE <222> (75) <223> Xaa is A or S <220> <221> UNSURE <222> (87) <223> Xaa is R or K <220> <221> UNSURE <222> (88) <223> Xaa is A or P <220> <221> UNSURE <222> (91) <223> Xaa is T or S <220> <221> UNSURE <222> (93) <223> Xaa is V or M <220> <223> Combined with BCMA's single flexible structure <400> 41 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Xaa Val Gln Xaa Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Xaa Xaa Glu Trp Val 35 40 45 Xaa Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Xaa Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Xaa Xaa Glu Asp Xaa Ala Xaa Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 42 <211> 126 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (11) <223> Xaa is S or L <220> <221> UNSURE <222> (14) <223> Xaa is P or A <220> <221> UNSURE <222> (27) <223> Xaa is F or V <220> <221> UNSURE <222> (44) <223> Xaa is E or G <220> <221> UNSURE <222> (49) <223> Xaa is A or S <220> <221> UNSURE <222> (75) <223> Xaa is A or S <220> <221> UNSURE <222> (87) <223> Xaa is R or K <220> <221> UNSURE <222> (88) <223> Xaa is A or P <220> <221> UNSURE <222> (93) <223> Xaa is V or M <220> <223> BCMA-binding single variable domain <400> 42 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Xaa Val Gln Xaa Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Xaa Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Xaa Arg Glu Gly Val 35 40 45 Xaa Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Xaa Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Xaa Xaa Glu Asp Thr Ala Xaa Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CD3-HCDR1 <400> 43 Thr Tyr Ala Met Asn 1 5 <210> 44 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CD3-HCDR2 <400> 44 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 45 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CD3-HCDR3 <400> 45 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CD3-LCDR1 <400> 46 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CD3-LCDR2 <400> 47 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CD3-LCDR3 <400> 48 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 49 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CD3-VH <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 50 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> CD3-VL <400> 50 Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 51 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> 1A10-V1 <400> 51 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Glu Pro Lys Ser 115 120 125 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 130 135 140 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 145 150 155 160 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 165 170 175 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 180 185 190 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 195 200 205 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 210 215 220 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 225 230 235 240 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 245 250 255 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 260 265 270 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 275 280 285 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 290 295 300 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 305 310 315 320 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 325 330 335 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 340 345 350 Ser Pro Gly Lys 355 <210> 52 <211> 1068 <212> DNA <213> Artificial Sequence <220> <223> 1A10-V1 <400> 52 60. caggtgcagc tggtggagtc cggcggcgga ctggtgcagc ctggaggatc tctgcggctg tcctgcgccg ccagcggata ctccagcaac gtggcttgca tggcctggta tcggcaggcc 120 cctggcaagg gcctggagtg ggtggctacc atcgtggctg acttcggcac caccaattat gccgctagcg tgaagggccg gttcaccatc agccaggata actccaagaa taccgtgtat ctgcagatga atagcctgcg ggccgaggat accgccgtgt attattgtgc cgctacccag 300 cggggcggca tcgactggtg tgatgagatc aactattggg gccagggcac cctggtgacc 360 gtgtcctccg gcgagcctaa gtcctccgac aagacccaca cctgccctcc ttgtcccgcc 420 cctgagctgc tgggcggacc atctgtgttc ctgttccctc ccaagccccaa ggacaccctg 480 atgatctcca ggacccctga ggtgacctgc gtggtggtgg atgtgtccca cgaggaccct 540 gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaatgctaa gaccaagccc cgggaggagc agtatacag cacctaccgg gtggtgagcg tgctgaccgt gctgcaccag 660 gattggctga atggcaagga gtacaagtgt aaggtgtcca acaaggctct gcctgccccc 720 atcgagaaga ccatctccaa ggccaagggc cagcctcggg agccccaggt gtataccctg 780 cccccctcca gggacgagct gaccaagaac caggtgagcc tgacctgtct ggtgaagggc 840 ttctatcctt ccgacatcgc cgtggagtgg gagtccaacg gccagcctga gaataactac 900 aagaccaccc cccctgtgct ggatagcgac ggctccttct tcctgtattc caagctgacc 960 gtggataagt cccggtggca gcagggcaat gtgttcagct gttccgtgat gcacgaggct 1020 ctgcacaacc actataccca gaagtccctg tccctgagcc ccggcaag 1068 <210> 53 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> 1A10-V2 <400> 53 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ser Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Glu Pro Lys Ser 115 120 125 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 130 135 140 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 145 150 155 160 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 165 170 175 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 180 185 190 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 195 200 205 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 210 215 220 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 225 230 235 240 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 245 250 255 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 260 265 270 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 275 280 285 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 290 295 300 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 305 310 315 320 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 325 330 335 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 340 345 350 Ser Pro Gly Lys 355 <210> 54 <211> 1068 <212> DNA <213> Artificial Sequence <220> <223> 1A10-V2 <400> 54 caggtgcagc tggtggagtc cggcggcgga ctggtgcagc ctggaggatc tctgcggctg 60 tcctgcgccg ccagcggata ctccagcaac gtggcttgca tggcctggta tcggcaggcc 120 cctggcaagg agcgggagtg ggtgagcacc atcgtggctg acttcggcac caccaattat 180 gccgctagcg tgaagggccg gttcaccatc agccaggata actccaagaa taccgtgtat 240 ctgcagatga atagcctgcg ggccgaggat accgccgtgt attattgtgc cgctacccag 300 cggggcggca tcgactggtg tgatgagatc aactattggg gccagggcac cctggtgacc 360 gtgtcctccg gcgagcctaa gtcctccgac aagacccaca cctgccctcc ttgtcccgcc 420 cctgagctgc tgggcggacc atctgtgttc ctgttccctc ccaagcccaa ggacaccctg 480 atgatctcca ggacccctga ggtgacctgc gtggtggtgg atgtgtccca cgaggaccct 540 gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaatgctaa gaccaagccc 600 cgggaggagc agtataacag cacctaccgg gtggtgagcg tgctgaccgt gctgcaccag 660 gattggctga atggcaagga gtacaagtgt aaggtgtcca acaaggctct gcctgccccc 720 atcgagaaga ccatctccaa ggccaagggc cagcctcggg agccccaggt gtataccctg 780 cccccctcca gggacgagct gaccaagaac caggtgagcc tgacctgtct ggtgaagggc 840 ttctatcctt ccgacatcgc cgtggagtgg gagtccaacg gccagcctga gaataactac 900 aagaccaccc cccctgtgct ggatagcgac ggctccttct tcctgtattc caagctgacc 960 gtggataagt cccggtggca gcagggcaat gtgttcagct gttccgtgat gcacgaggct 1020 ctgcacaacc actataccca gaagtccctg tccctgagcc ccggcaag 1068 <210> 55 <211> 359 <212> PRT <213> Artificial Sequence <220> <223> 1A11-V1 <400> 55 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ser Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Glu 115 120 125 Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 130 135 140 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 145 150 155 160 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 165 170 175 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 180 185 190 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 195 200 205 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 210 215 220 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 225 230 235 240 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 245 250 255 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 260 265 270 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 275 280 285 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 290 295 300 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 305 310 315 320 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 325 330 335 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 340 345 350 Leu Ser Leu Ser Pro Gly Lys 355 <210> 56 <211> 1077 <212> DNA <213> Artificial Sequence <220> <223> 1A11-V1 <400> 56 caggtgcagc tggtggagtc cggcggcgga ctggtgcagc caggaggatc tctgcggctg 60 tcctgtgccg cttccggctt caccttcaat tccgcttgca tgggctggtt caggcaggcc 120 cctggcaagg gcagggaggg agtgtccagg atcgagaccg gctacggcgg caccgtgtac 180 gctgacagcg tgaagggccg gttcaccatc agccgggata actccaagaa caccgtgtat 240 ctgcagatga atagcctgag ggctgaggat accgccgtgt actattgtgc tgctaagcgg 300 tcctggtgta cccccacctg gtggcacgag ctggactaca attactgggg ccagggcacc 360 caggtgaccg tgagctccgg cgagcctaag tcctccgaca agacccacac ctgccctcct 420 tgtcccgccc ctgagctgct gggcggacca tctgtgttcc tgttccctcc caagcccaag 480 gacaccctga tgatctccag gacccctgag gtgacctgcg tggtggtgga tgtgtcccac 540 gaggaccctg aggtgaagtt caactggtac gtggacggcg tggaggtgca caatgctaag 600 accaagcccc gggaggagca gtataacagc acctaccggg tggtgagcgt gctgaccgtg 660 ctgcaccagg attggctgaa tggcaaggag tacaagtgta aggtgtccaa caaggctctg 720 cctgccccca tcgagaagac catctccaag gccaagggcc agcctcggga gccccaggtg 780 tataccctgc ccccctccag ggacgagctg accaagaacc aggtgagcct gacctgtctg 840 gtgaagggct tctatccttc cgacatcgcc gtggagtggg agtccaacgg ccagcctgag 900 aataactaca agaccacccc ccctgtgctg gatagcgacg gctccttctt cctgtattcc 960 aagctgaccg tggataagtc ccggtggcag cagggcaatg tgttcagctg ttccgtgatg 1020 cacgaggctc tgcacaacca ctatacccag aagtccctgt ccctgagccc cggcaag 1077 <210> 57 <211> 359 <212> PRT <213> Artificial Sequence <220> <223> 1A11-V2 <400> 57 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Glu 115 120 125 Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 130 135 140 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 145 150 155 160 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 165 170 175 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 180 185 190 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 195 200 205 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 210 215 220 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 225 230 235 240 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 245 250 255 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 260 265 270 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 275 280 285 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 290 295 300 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 305 310 315 320 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 325 330 335 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 340 345 350 Leu Ser Leu Ser Pro Gly Lys 355 <210> 58 <211> 1077 <212> DNA <213> Artificial Sequence <220> <223> 1A11-V2 <400> 58 caggtgcagc tggtggagtc cggcggcgga ctggtgcagc caggaggatc tctgcggctg 60 tcctgtgccg cttccggctt caccttcaat tccgcttgca tgggctggtt caggcaggcc 120 cctggcaagg agagggaggg agtgtccagg atcgagaccg gctacggcgg caccgtgtac 180 gctgacagcg tgaagggccg gttcaccatc agccgggata actccaagaa caccgtgtat 240 ctgcagatga atagcctgaa ggctgaggat accgccgtgt actattgtgc tgctaagcgg 300 tcctggtgta cccccacctg gtggcacgag ctggactaca attactgggg ccagggcacc 360 caggtgaccg tgagctccgg cgagcctaag tcctccgaca agacccacac ctgccctcct 420 tgtcccgccc ctgagctgct gggcggacca tctgtgttcc tgttccctcc caagcccaag 480 gacaccctga tgatctccag gacccctgag gtgacctgcg tggtggtgga tgtgtcccac 540 gaggaccctg aggtgaagtt caactggtac gtggacggcg tggaggtgca caatgctaag 600 accaagcccc gggaggagca gtataacagc acctaccggg tggtgagcgt gctgaccgtg 660 ctgcaccagg attggctgaa tggcaaggag tacaagtgta aggtgtccaa caaggctctg 720 cctgccccca tcgagaagac catctccaag gccaagggcc agcctcggga gccccaggtg 780 tataccctgc ccccctccag ggacgagctg accaagaacc aggtgagcct gacctgtctg 840 gtgaagggct tctatccttc cgacatcgcc gtggagtggg agtccaacgg ccagcctgag 900 aataactaca agaccacccc ccctgtgctg gatagcgacg gctccttctt cctgtattcc 960 aagctgaccg tggataagtc ccggtggcag cagggcaatg tgttcagctg ttccgtgatg 1020 cacgaggctc tgcacaacca ctatacccag aagtccctgt ccctgagccc cggcaag 1077 <210> 59 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> A10-linker-A11-Fc <400> 59 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser 130 135 140 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val 145 150 155 160 Thr Phe Asn Ser Ala Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 165 170 175 Glu Arg Glu Gly Val Ala Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 195 200 205 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 210 215 220 Ala Met Tyr Tyr Cys Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp 225 230 235 240 Trp His Glu Leu Asp Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr 245 250 255 Val Ser Ser Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 260 265 270 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 275 280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 290 295 300 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 385 390 395 400 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 435 440 445 Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 <210> 60 <211> 1476 <212> DNA <213> Artificial Sequence <220> <223> A10-linker-A11-Fc <400> 60 caggtgcagc tggtggagag cggcggcgga tccgtgcagg ctggaggaag cctgcggctg 60 tcctgtgccg cctccggata ctcctccaac gtggcctgta tggcttggta caggcaggct 120 cctggcaagg agcgggagtg ggtggctacc atcgtggccg acttcggcac caccaattac 180 gctgctagcg tgaagggcag gttcaccatc agccaggata acgctaagaa taccgtgtac 240 ctgcagatga atagcctgaa gcccgaggac tccgctatgt actattgtgc cgctacccag 300 aggggcggca tcgattggtg cgacgagatc aattattggg gccagggcac cctggtgacc 360 gtgagcagcg gcggaggcgg aagcggagga ggaggaagcc aggtgcagct cgtggagtcc 420 ggcggcggaa gcgtgcaggc tggtggaagc ctgaggctgt cctgtgctgc tagcggcgtg 480 accttcaact ccgcttgtat gggctggttc cggcaggctc ccggcagga gagagaggggc 540 gtggccagga tcgagaccgg ctatggcggc accgtgtacg ccgacagcgt gaagggacgg 600 ttcaccatct ccaggataa cgctaaaaac accgtgtatc tgcagatgaa cagcctgaag 660 ccagaggata ccgctatgta ttactgtgct gccaagcgga gctggtgtac ccctacctgg 720 tggcacgagc tggactacaa ctattggggc cagggaaccc aggtgaccgt gtccagcggc 780 gagcccaagt ccagcgacaa gacccacc tgtcctcctt gccctgctcc agaggccgct 840 ggcggaccta gcgtgttcct gttccctcct aagcctagg acacctgat gatctccaga 900 acacctgaag tgacctgcgt gttcgtggat gttctcacg aggaccctga ggtgaagttc 960 aactggtacg tggacgggt ggaagtgcac aacgccaaga ccaagccctcg ggaggacag 1020 tacaactcca cctacagagt gtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 1080 ggaaaaggt acagtgcaa ggtgtccaac aaggccctgg ccgctcctat cgagaagact 1140 atctccaagg ctaagggcca gcctagagag cctcaagtgt gcaccctgcc tccatctcgg 1200 gaagagatga ccaagaacca ggtgtctctg tcttgcgccg tcaagggctt ctacccttcc 1260 gacatcgccg tggaatggga gtctaacggc cagcctgaga acaactacaa gaccacccct 1320 cctgtgctgg actctgacgg ctccttcttc ctcgtgtcca agctgaccgt ggacaagtct 1380 cggtggcagc agggcaacgt gttctcctgc tctgtgatgc acgaggctct gcacaaccac 1440 tacacccaga agtccctgtc cctgtcccct ggcaag 1476 <210> 61 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> anti-CD3-HC-Fc <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys 355 360 365 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 62 <211> 1365 <212> DNA <213> Artificial Sequence <220> <223> anti-CD3-HC-Fc <400> 62 gaggtgcagc tggtggaatc tggcggaggc ctcgtgcagc ccggcggctc tctgagactg 60 tcttgcgccg cttctggctt taccttcaat acctatgcca tgaattgggt gcggcaggcc 120 cctggcaagg gcctggaatg ggtcgccaga atcagatcca agtacaacaa ctacgccacc 180 tactacgccg attctgtgaa agacagattc accatctctc gggacgactc taagaacacc 240 gcctacctgc agatgaacaa cctgaagacc gaggacaccg ctatgtacta ctgcgtgaga 300 cacggcaact tcggcaactc ctacgtgtcc tggttcgcct actggggcca gggcaccctg 360 gtcacagttt cttctgcctc taccaagggc ccttccgtgt tccctctggc ccctagcagc 420 aaaagcacct ccggcggaac cgccgccctg ggctgtctgg tgaaggacta cttccccgag 480 cctgtgacgg tgtcttggaa ctccggcgct ctgacctctg gcgtgcatac ctttcctgcc 540 gtgctgcagt ccagcggact gtactccctg tcctccgtcg tgacagttcc ctcctctagc 600 ctgggcacac agacctacat ctgcaacgtg aaccacaagc cttctaacac caaagtggac 660 aagaaggtgg aacccaaatc ctgcgacaag acacacacct gtcctccttg ccctgctcct 720 gaggccgctg gcggccctag cgtgtttctg tttcctccta agcccaagga cacactgatg 780 atctccagaa ccccagaagt gacctgcgta gtggtggacg tgtcccacga ggatcctgag 840 gtgaagttca actggtacgt ggatggcgtg gaagtgcaca acgccaagac caagcctaga 900 gaagagcagt acaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggac 960 tggctgaacg gcaaagagta caagtgcaag gtgtccaaca aggccctggc tgctcctatc 1020 gagaagacca tctccaaggc taagggacag cctcgggaac ctcaagtgta caccctgcct 1080 ccttgtagag aggaaatgac caagaaccag gtgtctctgt ggtgcctggt caagggcttc 1140 tacccttctg acatcgccgt ggagtgggag tccaacggcc agcctgagaa caactacaaa 1200 accacccctc cagtgctgga ctccgacggc tccttcttcc tgtactccaa gctgacagtg 1260 gataagtcta gatggcagca gggcaatgtg ttctcctgct ccgtgatgca cgaggctctg 1320 cacaaccggt tcacccagaa gtccctgtcc ctgtcccctg gcaag 1365 <210> 63 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> anti-CD3-LC <400> 63 Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 64 <211> 645 <212> DNA <213> Artificial Sequence <220> <223> anti-CD3-LC <400> 64 gagctggtgg tgacccagga gccatccctg accacaagcc caggaggaac cgtgacactg 60 acctgtagat ccagcaccgg cgccgtgacc acaagcaact acgctaattg ggtgcagcag 120 aagcctggac aggctccaag gggactgatc ggaggaacca acaagagagc ccccggaaca 180 ccagctcggt tttccggcag cctgctggga ggcaaggccg ctctgacaat caccggcgtg 240 cagccagagg acgaggccga gtactattgc gctctgtggt attctaatct gtgggtgttt 300 ggaggaggaa caaagctgac cgtgctggga cagcccaagg ctaaccctac agtgaccctg 360 ttcccccctt cttccgagga gctgcaggcc aataaggcca ccctggtgtg cctgatctct 420 gacttttacc caggagctgt gacagtggct tggaaggctg atggctcccc tgtgaaggct 480 ggcgtggaga ccacaaagcc aagcaagcag tctaacaata agtacgccgc tagctcttat 540 ctgtctctga cccccgagca gtggaagtcc cacaggtctt attcctgcca ggtgacacat 600 gagggcagca cagtggagaa gaccgtggcc cctacagagt gttct 645 <210> 65 <211> 492 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-A10-v1-linker-A11-v1-Fc <400> 65 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 145 150 155 160 Thr Phe Asn Ser Ala Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 165 170 175 Gly Arg Glu Gly Val Ser Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 195 200 205 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp 225 230 235 240 Trp His Glu Leu Asp Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr 245 250 255 Val Ser Ser Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 260 265 270 Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 275 280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 290 295 300 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 385 390 395 400 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 435 440 445 Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 <210> 66 <211> 1476 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-A10-v1-linker-A11-v1-Fc <400> 66 caagtgcagc tggtcgagtc cggcggcgga ctggtgcagc caggcggctc cctccggctg 60 tcctgtgccg cctccggcta ctcctctaac gtggcctgca tggcctggta tagacaggct 120 cctggcaagg gcctggaatg ggtggccacc attgtggccg acttcggcac caccaactac 180 gccgcttccg tcaagggcag attcaccatc tcccaggata actccaagaa caccgtgtac 240 ctgcagatga actccctgag ggctgaagat accgctgtgt actactgtgc tgccacacag 300 agaggcggca tcgattggtg cgacgagatc aactattggg gacagggcac tttggtgacc 360 gtgtccagcg gaggcggcgg ctccggcggg ggcggctctc aggttcagct ggtcgagtcc 420 ggcggtggcc tggtgcagcc cggcggaagc ctgagactgt cctgcgccgc ctctggcttt 480 accttcaact ctgcctgtat gggctggttt cggcaagccc ctgggaaagg cagagagggc 540 gtgagccgga tcgagacagg ctacggcggc acagtttacg ctgactctgt gaagggaaga 600 ttcaccatca gccgggacaa ttccaaaaat accgtgtacc tgcagatgaa cagcctgcgg 660 gccgaggaca cagctgtcta ctactgcgcg gccaagcggt cttggtgtac ccctacctgg 720 tggcacgagc tggactacaa ctactggggc cagggaactc aggtgaccgt gtcctccggc 780 gagcccaagt cctctgacaa gacacacacc tgtcctccat gccctgctcc tgaggccgct 840 ggcggacctt ccgtgttcct gtttcctcct aagcccaagg acaccctgat gatctctcgg 900 acccctgagg tgacctgcgt ggtggtggac gtgtctcatg aggatcctga agtgaagttc 960 aactggtacg tggatggcgt ggaagtgcac aacgccaaga ccaagcctag agaagaacag 1020 tacaactcca cctacagagt ggtcagcgtg ctgaccgtgc tgcatcagga ctggctgaac 1080 ggaaaagagt acaagtgcaa ggtgtccaac aaggctctgg ctgcccctat cgagaagacc 1140 atctccaagg ccaagggcca gccccgcgag cctcaggtgt gcaccctgcc tccatctaga 1200 gaagagatga ccaagaacca ggtctccctc agctgcgccg tgaagggctt ctacccttct 1260 gacatcgctg tggaatggga aagcaatggc caacccgaga acaactacaa gaccaccccc 1320 cctgtgctgg actccgacgg cagcttcttc ctggtgtcaa agctgacagt ggacaagtcc 1380 agatggcagc agggcaacgt gttcagctgc agtgtgatgc acgaggccct gcacaaccac 1440 tacacccaga aatctctgtc tctgtctcct ggcaaa 1476 <210> 67 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> anti-CD3-scFv-Fc <400> 67 Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg 165 170 175 Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp 180 185 190 Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln 195 200 205 Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Val Arg 210 215 220 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Glu Pro Lys Ser Ser Asp 245 250 255 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 260 265 270 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 275 280 285 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 290 295 300 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 305 310 315 320 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 325 330 335 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 340 345 350 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro Ile Glu 355 360 365 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 370 375 380 Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 385 390 395 400 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 405 410 415 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 420 425 430 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 435 440 445 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 450 455 460 Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro 465 470 475 480 Gly Lys <210> 68 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> anti-CD3-scFv-Fc <400> 68 gagctggtcg tcacccaaga gccctctctg accacaagcc ctggcggaac cgtgaccctg 60 acctgtaggt cctccaccgg cgctgtgacc acctcgaact atgccaactg ggtgcaacag 120 aagcccggcc aggcacccag aggcctgatc ggcggcacca acaagcgcgc ccccggcaca 180 cctgccagat tctctggctc tctgctgggc ggaaaggccg ccctgaccat caccggcgtc 240 cagcccgagg acgaggccga gtactactgc gctctgtggt actccaacct gtgggtgttt 300 ggcggcggca ccaagctgac cgtgctgggc ggcggtggct ctggaggagg cggatctggt 360 ggcggaggct ctgaagtgca gctcgttgag agcggcggcg gcctggtgca gcctggcggc 420 tccctgagac tgtcttgcgc cgcttccggc ttcaccttca acacctatgc catgaattgg 480 gtccggcagg cccccggcaa gggactggaa tgggtggcta gaatcagatc caagtacaac 540 aactacgcta cctactacgc cgactctgtg aaggaccggt tcaccatctc tcgggacgac 600 tccaagaaca ccgcctacct gcagatgaac aatctgaaga ccgaggatac cgctatgtac 660 tactgtgtgc ggcacggcaa cttcggcaac tcctacgtgt cctggttcgc ctactggggc 720 cagggcactc tcgtgaccgt gtccagcggc gagcccaagt cctctgacaa gacccacacc 780 tgccctccat gtcctgctcc tgaggctgct ggcggccctt ccgtgtttct gttccctcct 840 aagcctaaag atacactgat gatctcccgg acccctgaag ttacatgcgt ggtggtggac 900 gtgtctcacg aggaccccga agtgaagttc aactggtatg tggatggcgt ggaagtgcac 960 aatgccaaaa ccaagcctcg ggaggaacag tacaactcta cctacagagt ggtaagcgtg 1020 ctgaccgtgc tgcaccagga ctggctgaac ggcaaagagt acaagtgcaa ggtgtctaac 1080 aaggctctgg ccgcccctat cgagaagaca atttccaagg ccaagggcca gcctagagaa 1140 cctcaggtgt acaccctgcc tccttgcaga gaagagatga ccaagaacca ggttagccta 1200 tggtgcctgg tgaagggttt ctacccttct gacatcgccg tggagtggga gtccaatggc 1260 caaccagaga acaactacaa gacaacacca cctgtgctgg actccgatgg ctccttcttc 1320 ctgtactcta agctgacagt ggacaagtcc agatggcagc agggcaacgt gttctcctgc 1380 tccgtgatgc atgaagctct gcacaaccgg ttcacccaga aatccctgag cctgtctcct 1440 ggaaaa 1446 <210> 69 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-A10-v1-Fc <400> 69 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Glu Pro Lys Ser 115 120 125 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 130 135 140 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 145 150 155 160 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 165 170 175 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 180 185 190 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 195 200 205 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 210 215 220 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro 225 230 235 240 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 245 250 255 Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 260 265 270 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 275 280 285 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 290 295 300 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 305 310 315 320 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 325 330 335 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 340 345 350 Ser Pro Gly Lys 355 <210> 70 <211> 1068 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-A10-v1-Fc <400> 70 caagtccagc tggttgagtc cggcggaggc ctggtgcagc caggcggctc cctgagactg 60 tcttgtgctg cctctggcta ctcctctaat gtggcctgca tggcctggta ccggcaggcc 120 cctggcaagg gcctggaatg ggtggctact atcgtggccg acttcggcac caccaattat 180 gctgcctctg tgaagggacg gttcaccatc tcccaggaca actccaagaa caccgtgtac 240 ctgcagatga acagcctgcg ggccgaggac acagccgtgt actactgtgc cgctacacag 300 agaggcggca tcgactggtg cgacgagatc aactactggg gccaaggaac cctggtgaca 360 gtgtcttctg gcgagcccaa atcctccgat aaaacacaca cctgtcctcc ttgccctgct 420 cccgaggccg ccggcggccc tagcgtattc ctgtttcccc ctaagcctaa ggacacattg 480 atgatctctc ggacacccga agtgacctgc gtggtcgtgg acgtgtctca cgaagatcct 540 gaggtgaagt tcaactggta tgtggatggc gtggaagtgc ataacgccaa gaccaagccc 600 agagaggaac agtacaactc tacctacaga gtggtgtccg tcctgaccgt gctgcatcag 660 gattggctga acggcaaaga gtacaagtgc aaggtgtcta acaaggctct ggccgctccc 720 attgagaaga ccatctccaa ggctaagggt cagcctagag agcctcaagt gtgcaccctg 780 cctccatcta gagaagagat gaccaagaac caggtcagcc tgtcttgtgc agtgaaaggc 840 ttctacccaa gcgacatcgc cgtggaatgg gagtctaatg gccagcctga aaacaactac 900 aagaccaccc ctcctgtgct cgactccgac ggctccttct tcctggtgtc caagctgacc 960 gtggacaagt cccgctggca gcagggcaac gtgttctcct gctccgtgat gcacgaggct 1020 ctgcacaacc actacaccca gaagagcctg agcctgtccc ctggaaag 1068 <210> 71 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-A11-v1-linker-anti-CD3-scFv-Fc <400> 71 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ser Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Thr Ser 130 135 140 Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val 145 150 155 160 Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala 165 170 175 Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro 180 185 190 Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile 195 200 205 Thr Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp 210 215 220 Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 245 250 255 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 260 265 270 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala 275 280 285 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 290 295 300 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 305 310 315 320 Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala 325 330 335 Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr 340 345 350 Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala 355 360 365 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Glu Pro Lys 370 375 380 Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 385 390 395 400 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 405 410 415 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 420 425 430 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 435 440 445 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 450 455 460 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 465 470 475 480 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala 485 490 495 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 500 505 510 Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln 515 520 525 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 530 535 540 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 545 550 555 560 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 565 570 575 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 580 585 590 Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser 595 600 605 Leu Ser Pro Gly Lys 610 <210> 72 <211> 1839 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-A11-v1-linker-anti-CD3-scFv-Fc <400> 72 caggtgcagc tcgtggaatc cggcggcggc ctggtgcagc ctggcggctc tctgagactg 60 tcttgtgctg cctctggctt tactttcaac tccgcctgca tgggctggtt cagacaggcc 120 cccggaaagg gccgggaggg cgtgtctcgg atcgagacag gctacggcgg taccgtgtac 180 gccgactctg tgaaaggcag attcaccatc tccagagaca actccaagaa taccgtctac 240 ctgcagatga atagcctccg cgccgaggac accgctgtgt actactgcgc cgctaagaga 300 tcttggtgca cccccacctg gtggcacgag ctggactaca actactgggg ccagggaacc 360 caggtgaccg tgtccagcgg cggaggcggc tctgaactgg tggtgaccca agagccttcc 420 ctgaccacct cccctggcgg cacagtgaca ctgacctgta gatcctccac cggcgccgtg 480 accacctcta actatgctaa ctgggtgcag cagaaacctg gccaggcacc tcggggactg 540 atcggcggca ccaacaagcg ggctcctgga acacctgcta gattctccgg ctccttgctg 600 ggcggcaagg ccgccctgac catcaccggg gtgcaacctg aggacgaggc tgagtattac 660 tgcgctctgt ggtactccaa cctgtgggtc tttggcggcg gaaccaagct gaccgtgctg 720 ggcggcggcg gctccggcgg aggcggctcc ggaggaggcg gcagcgaggt gcagctggtc 780 gagtctggcg gcggcctggt gcagcccggc ggctcgctgc ggctgagctg tgccgcctcc 840 ggcttcacat tcaataccta cgccatgaac tgggtcaggc aggctcctgg caaaggcctg 960. sightgggtgg sightccg sightly aacaactatg sightly cgccgattct gtgaaggaca gattcacaat ctcccgggac gactctaaga acaccgctta cctgcagatg aacaacctga agaccgagga tacagctatg tactactgtg tgcggcacgg caacttcggc aattcctacg tgtcctggtt cgcctactgg ggacaaggca ccttggtgac agtttctagc 1140 ggcgagccca agtcttctga caagacacac acctgccctc cttgtcctgc ccctgaggcc gctggcggac cttctgtgtt tctgtttcca cctaagccta aggataccct gatgatctct agaacccccg aggtgacctg cgtggtcgtg gacgtgagcc atgaagatcc tgaagtgaag ttcaactggt atgtggacgg cgtggaagtg cacaatgcca agaccaagcc tagagaagag cagtacaact ctacctacag agtggtgtcc gtgctgaccg ttctgcatca ggactggctg aacggcaaag agtacaagtg caaggtgtct aacaaggctc tggctgcccc tatcgagaag accatttcca aggccaaggg ccagccacgc gaacctcaag tgtacaccct gccaccttgc agagaggaga tgaccaagaa ccaggtgagc ctgtggtgcc tggtgaaagg cttctacccc 1620 agcgacatcg ccgtggaatg ggagtccaac ggccagcccg agaacaacta caaaaccacc 1680 cctcctgtgc tggattctga tggttccttc ttcctgtact ctaagctgac cgtggacaag 1740 tcccggtggc agcagggcaa cgtgttcagc tgctccgtga tgcacgaagc cctgcacaac 1800 cggttcaccc agaaatccct gtctctgtct ccaggcaag 1839 <210> 73 <211> 359 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-A11-v1-Fc <400> 73 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Ala 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ser Arg Ile Glu Thr Gly Tyr Gly Gly Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Arg Ser Trp Cys Thr Pro Thr Trp Trp His Glu Leu Asp 100 105 110 Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Glu 115 120 125 Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 130 135 140 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 145 150 155 160 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 165 170 175 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 180 185 190 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 195 200 205 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 210 215 220 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 225 230 235 240 Ala Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 245 250 255 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 260 265 270 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 275 280 285 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 290 295 300 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 305 310 315 320 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 325 330 335 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 340 345 350 Leu Ser Leu Ser Pro Gly Lys 355 <210> 74 <211> 1077 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-A11-v1-Fc <400> 74 caggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggaggcag cctgcggctg 60 tcgtgcgctg cctccggatt caccttcaac tccgcctgta tgggctggtt cagacaggcc 120 cctggcaagg gtcgcgagg cgttagccgg atcgaaacag gctacggcgg aaccgtctac 180 gccgactccg tgaaaggcag attcaccatc tctcgggaca attctaagaa taccgtgtac 240 ctgcagatga actctctgag ggccgaggac accgctgtgt actactgcgc cgctaaacgg 300 tcctggtgca ctcctacctg gtggcatgag ctggactaca attattgggg ccaaggaaca 360 caagtgacag tatcctctgg cgagcccaag tcctccgata aaacccacac ctgccctcct 420 tgtcctgctc cagaggccgc tggcggccct tccgtgtttc tgtttccacc taagcccaag 480 gacacacactga tgatcagcag aacccctgaa gtcacctgtg tggtcgtgga cgtgagccat 540 gaagatcctg aggtgaagtt caactggtac gtggacggcg tggaagtgca caacgctaaaa 600 accaagccta gagaggaaca gtacaactct acctacagag tggtgtccgt gctgacagtg 660 ctgcaccagg actggctgaa cggcaaagag tacaagtgca aggtgagtaa caaggccctg 720 gctgctccca tcgagaagac catctccaag gccaagggcc agcctcggga gcctcaggtg 780 tgcaccctgc ctccatctag agaagagatg accaagaacc aggtgtccct gtcttgtgcc 840 gtcaagggct tctacccttc tgacatcgcc gtggaatggg agtctaacgg ccagcccgag 900 aacaactata agaccacccc ccccgtgctg gattctgatg gctccttctt cctcgtgtcc 960 aagctgaccg tggacaagtc cagatggcag cagggcaacg tgttctcctg ctctgtgatg 1020 cacgaggcac tgcacaacca ctacacccag aagtccctta gcctgtctcc tggcaag 1077 <210> 75 <211> 343 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-A10-v1-linker-anti-CD3-VL <400> 75 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ser Asn Val Ala 20 25 30 Cys Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Val Ala Asp Phe Gly Thr Thr Asn Tyr Ala Ala Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Gln Arg Gly Gly Ile Asp Trp Cys Asp Glu Ile Asn Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Glu Leu Val Val Thr Gln Glu Pro Ser Leu Thr Thr Ser Pro Gly Gly 130 135 140 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 145 150 155 160 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 165 170 175 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 180 185 190 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Val 195 200 205 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 210 215 220 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 225 230 235 240 Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 245 250 255 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 260 265 270 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala 275 280 285 Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 290 295 300 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 305 310 315 320 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 325 330 335 Val Ala Pro Thr Glu Cys Ser 340 <210> 76 <211> 1029 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-A10-v1-linker-anti-CD3-VL <400> 76 caagtgcagc tggtggaatc tggcggcgga ctggtgcagc caggcggctc cctgagactg 60 tcctgtgctg cttctggcta ctcgtccaac gtggcctgca tggcctggta tcgccaggct 120 cctggcaaag gcctggaatg ggtggctacc atcgtggccg acttcggcac tacaaactac 180 gccgcctccg tgaagggcag attcaccatc tctcaggaca actccaagaa caccgtgtac 240 ctgcagatga acagcctgcg ggctgaggac accgccgtgt actactgtgc cgctacccag 300 cggggaggca tcgactggtg cgacgagatc aactactggg gccagggaac cttggttaca 360 gtgtccagcg gcggaggcgg ctccgaactg gtggtgaccc aagagccttc tctgaccacc 420 tcccctggcg gcacagtgac cctgacctgt agatcctcta ccggtgccgt gacaacctct 480 aattacgcca actgggtcca gcagaagccc ggccaagccc ctagaggcct gatcggcggg 540 acgaacaagc gggctcctgg cacccctgcc cggttcagcg gctctctgct gggcggcaag 600 gctgccctga caatcaccgg cgtgcagcct gaggatgagg ccgagtacta ctgcgctctg 660 tggtatagca acctgtgggt gttcggagga ggcacaaagc tgaccgtgct gggccagcct 720 aaggccaatc ctaccgtgac cctgtttcct ccatcttctg aagagctgca ggccaacaaa 780 gccaccctcg tgtgcctgat ctccgacttc taccccggcg ctgtgaccgt ggcatggaag 840 gccgatggct cccctgtgaa agctggcgtc gaaaccacca agcctagcaa gcagtccaac 900 aacaagtacg ctgcctcttc ctacctgtct ctgacccccg agcagtggaa gtcccataga 960 agctactctt gccaggtgac ccacgagggc tccaccgtcg agaagaccgt ggcccccacc 1020 gagtgctcc 1029 <210> 77 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-HC-Fc <400> 77 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 78 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-HC-Fc <400> 78 gaggtgcagc tgctggaatc tggaggcggc ctggtgcaac ccggcggatc tctgagactg 60 tcttgtgccg cttctggctt caccttttcc tcctacgcca tgtcctgggt cagacaggcc 120 cctggaagg gactggaatg ggtgtctgcc atctccggct ctggcggctc tacatactac gccgacagcg tgaagggcag attcaccatc agccgggaca actccaagaa caccctctac ctgcagatga actctctgcg ggccgaggat accgccgtgt actactgcgc caaagtgctg ggatggttcg actactgggg ccaaggcacg ctcgtgaccg tgtcctccgc ctccaccaaa ggaccttccg tgtttcctct ggccccttcc cctccggcgg taccgctgcc 420 ctgggctgcc tggtcgagga ctacttccct gagcccgtga ccgtgtcttg gaactccggc 480 gctctgacct ctggcgtgca caccttcccc gctgttctgc agtccagcgg cctgtactcc 540 ctgtcctctg tggtgacagt gcctagctct tctctgggca cccagaccta catctgcaac 600 gtgaaccaca agccctccaa caccaaggtg gatgagaaag tggaacccaa gtcttgcgac aagacacata cctgtcctcc ttgccctgct cctgaggccg ctggcggccc ttctgtgttc 720 ctgttcccac ccaagcctaa ggacaccctg atgatctctc ggacccctga ggtcacctgc 780 gtggtcgtgg acgtgtccca tgaagatcct gaagtgaagt tcaactggta cgtggatggc 840 gtggaagtgc acaacgccaa gaccaagcct cgggaagagc agtacaactc cacatacaga 900 gtggtgagcg tgctgaccgt gctgcaccag gactggctga acggcaaaga gtacaagtgc 960 aaggtgtcca acaaggctct cggcgccccc atcgagaaga caatctccaa ggccaagggc 1020 cagcctagag agcctcaggt gtgcaccctg cctccatctc gcgacgagct gactaagaac 1080 caggtgtccc tgtcctgtgc tgtgaaaggc ttctaccctt ctgacatcgc cgtggagtgg 1140 gagtctaacg gccagcccga gaacaactac aagaccacac ctcctgttct ggactccgac 1200 ggctccttct tcctggtgtc taagctgacc gtggacaagt ccagatggca gcagggcaac 1260 gtgttctcct gctccgtgat gcacgaggct ctgcacaaca gattcaccca gaagtctctc 1320 agcctgtccc ctggcaag 1338 <210> 79 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-LC <400> 79 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Tyr Pro Pro 85 90 95 Asp Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 80 <211> 648 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-LC <400> 80 gagatcgtgc tgacccagtc tcctggaaca ctgtctctgt cccctggcga gagagctacc 60 ctgagctgca gagcctctca gtccgtgtcc tcctcctacc tggcctggta ccagcagaag 120 cctggccagg cccctcggct gctgatctac ggcgctagct ctcgggccac cggcatccct 180 gacagattct ccggctccgg ctctggcaca gattttaccc tgaccatctc cagactggaa 240 cctgaggact tcgccgtgta ctactgccag cagtacggct acccccccga cttcaccttc 300 ggccaaggca ccaaagtgga aatcaagcgg accgtggctg ctccttctgt gttcatcttc 360 cctccttccg accggaagct gaagtctgga accgcctccg tggtgtgcct gctgaacaac 420 ttctacccta gagaggccaa ggtgcagtgg aaggtggaca acgccctgca gagcggcaac 480 tcccaagagt ccgtcaccga gcaggactct aaggactcca cctactccct gtcctctacc 540 ctgacactgt ccaaggccga ctacgagaag cacaaggtgt acgcctgcga ggtgacccac 600 cagggcctgt cttctcctgt gaccaagtcc ttcaacagag gcgaatgt 648 <210> 81 <211> 671 <212> PRT <213> Artificial Sequence <220> <223> anti-BCMA-VH-anti-CD3-VL-Fc <400> 81 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu 225 230 235 240 Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr 245 250 255 Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro 260 265 270 Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro 275 280 285 Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala 290 295 300 Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys 305 310 315 320 Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu 325 330 335 Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 340 345 350 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 355 360 365 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 370 375 380 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 385 390 395 400 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 405 410 415 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 420 425 430 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 435 440 445 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 450 455 460 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 465 470 475 480 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 485 490 495 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 500 505 510 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 515 520 525 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 530 535 540 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 545 550 555 560 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 565 570 575 Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 580 585 590 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 595 600 605 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 610 615 620 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 625 630 635 640 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 645 650 655 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665 670 <210> 82 <211> 2013 <212> DNA <213> Artificial Sequence <220> <223> anti-BCMA-VH-anti-CD3-VL-Fc <400> 82 gaggtgcagc tgctggaatc aggcggaggc ctcgtgcagc caggtggcag cctgcggctg 60 tcttgtgccg cctctggctt caccttctcc tcctacgcca tgtcctgggt gcggcaggcc 120 cctggcaagg gactggagtg ggtctccgcc atctctgggt ccggaggctc cacctactac 180 gctgattctg tgaaaggcag attcaccatc tctcgggaca actccaagaa cacactgtac 240 ctgcagatga actccctgag agccgaggac accgccgtgt actactgcgc caaggtgctg 300 ggatggttcg actactgggg ccagggcacc ttagtgaccg tgtcaagcgc cagcacaaag 360 ggacctagcg tatttcctct ggcccctagc tctaagtcca cctctggtgg caccgcagcc 420 ctgggctgtc tggtggaaga ctacttccct gaacccgtga cagtgtcttg gaactccggc 480 gctctgacat ctggcgtgca caccttccct gctgttcttc aatcttccgg cctgtacagc 540 ctgtcctctg ttgtcacagt gcccagttct tctctgggca cccagacata catctgtaac 600 gtgaaccaca agccttccaa caccaaagtg gacgagaaag tggaacctaa gtcctgcgat 660 ggcggcggcg ggtccggtgg agggggatct caggctgtgg tcacccaaga gccctccctg 720 accgtgtccc ctggcggcac cgtgacactg acctgcggct cttccacagg cgccgtgacc 780 acctccaact acgccaactg ggtgcaagag aagcctggcc aggccttcag aggcctgatc 840 ggcggaacca acaagcgggc tcctggcacc cctgccagat tctctggaag tctgctgggt 900 ggcaaggctg cactgaccct gtccggcgct caacctgagg atgaggccga gtactactgc 960 gccctgtggt attccaacct gtgggtcttc ggcggtggca ccaagctgac cgtgctgtct 1020 tctgcctcca ccaaaggccc ctcggtgttc cctctggctc ctagttccaa gtctacctcc 1080 ggcggtacag ctgctctggg ctgcctggtc aaggattact tcccagagcc tgtgaccgtg 1140 tcttggaact cgggcgccct gacttctgga gtgcacacat tccctgctgt gctgcagtcc 1200 tctggcctgt actctctgtc ttcggtcgtg acagtgcctt ccagcagcct cggcacacag 1260 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggataa gaaggtggaa 1320 cccaaatctt gcgacaagac ccacacctgt cctccttgtc ctgcccctga ggccgctggc 1380 ggcccttctg tgttcctgtt cccgccaaag cctaaggaca cgctgatgat ctcccggacc 1440 cctgaggtga cctgcgtggt ggtggatgtg tctcatgagg accctgaagt gaagttcaac 1500 tggtacgtgg acggcgtcga ggtgcacaac gccaaaacca agcccagaga ggaacagtac 1560 aacagcacct acagagtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 1620 aaagagtaca agtgcaaggt gagcaataag gccctgggcg cccctatcga gaagaccatc 1680 tccaaggcca agggccagcc tagagaacct caggtgtaca ccctgcctcc ttgcagagac 1740 gagctgacca agaaccaggt ctctctgtgg tgcctggtga agggcttcta cccttctgac 1800 atcgccgtgg aatgggagtc caatggacag cccgagaaca actacaagac cacacctcca 1860 gtgctggact ccgacggctc cttcttcctg tacagcaagc tgacagtgga caagtccaga 1920 tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggctctgca caaccactac 1980 acccagaagt ccctgtccct gtctcctgga aag 2013 <210> 83 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> anti-CD3-VH-CL <400> 83 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val 115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 84 <211> 696 <212> DNA <213> Artificial Sequence <220> <223> anti-CD3-VH-CL <400> 84 gaggtgcagc tgctggaatc cggcggagga ctggtccagc ctggcggctc tctgcggctg 60 tcttgtgctg cttctggctt caccttctcc acatatgcca tgaactgggt cagacaggcc 120 cccggcaagg gcctggaatg ggtgtccaga atcagatcca agtacaacaa ctacgccacc 180 tactacgccg attccgtgaa gggacggttc accatctccc gggacgactc caagaacaca 240 ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgcgtgcgg 300 cacggcaact tcggcaattc ctacgtgtcc tggttcgcct actggggcca gggcaccctg 360 gtcaccgtgt cttccgcctc tgtggccgct ccttctgtgt tcatcttccc tccttccgac 420 gagcagctga agtctggcac agcttctgtg gtgtgcctgc tgaacaactt ctaccctaga 480 gaggccaagg tgcagtggaa ggtggacaac gccctgcagt ccggcaactc ccaagagtcc 540 gtgaccgagc aggactctaa ggactctacc tactccctgt cctccaccct gaccctgagc 600 aaggccgact acgagaagca caaagtgtac gcctgcgaag tgacccacca gggcctgtcc 660 tctcctgtga ccaagtcctt caacagaggc gagtgc 696

Claims

1. A multispecific antibody comprising (i) The first antigen binding module that binds to the first antigen; (ii) the second antigen-binding module that binds to the second antigen; and, (iii) The third antigen-binding module that binds to the first antigen; Wherein the first antigen is BCMA and the second antigen is CD3, both the first antigen-binding module and the third antigen-binding module are single variable domains. The first antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 10, CDR2 of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 of the amino acid sequence shown in SEQ ID NO: 12; and the third antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 13, CDR2 of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 of the amino acid sequence shown in SEQ ID NO: 15; or, the third antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 10, CDR2 of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 of the amino acid sequence shown in SEQ ID NO: 12, and the first antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 13, CDR2 of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 of the amino acid sequence shown in SEQ ID NO:

15. Furthermore, the second antigen-binding module comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises: HCDR1 with the amino acid sequence shown in SEQ ID NO: 43, HCDR2 with the amino acid sequence shown in SEQ ID NO: 44, and HCDR3 with the amino acid sequence shown in SEQ ID NO:

45. The light chain variable region comprises: LCDR1 with the amino acid sequence shown in SEQ ID NO: 46, LCDR2 with the amino acid sequence shown in SEQ ID NO: 47, and LCDR3 with the amino acid sequence shown in SEQ ID NO:

48.

2. The multispecific antibody according to claim 1, wherein the first antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 10, CDR2 of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 of the amino acid sequence shown in SEQ ID NO: 12, and the third antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 13, CDR2 of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 of the amino acid sequence shown in SEQ ID NO:

15.

3. The multispecific antibody according to claim 1, wherein the third antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 10, CDR2 of the amino acid sequence shown in SEQ ID NO: 11, and CDR3 of the amino acid sequence shown in SEQ ID NO: 12, and the first antigen-binding module comprises the following complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO: 13, CDR2 of the amino acid sequence shown in SEQ ID NO: 14, and CDR3 of the amino acid sequence shown in SEQ ID NO:

15.

4. The multispecific antibody according to claim 2, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

41.

5. The multispecific antibody according to claim 4, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 21, 37 or 38.

6. The multispecific antibody according to claim 2, wherein the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

42.

7. The multispecific antibody according to claim 6, wherein the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 23, 39 or 40.

8. The multispecific antibody according to claim 3, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

42.

9. The multispecific antibody according to claim 8, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 23, 39 or 40.

10. The multispecific antibody according to claim 3, wherein the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

41.

11. The multispecific antibody of claim 10, wherein the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 21, 37 or 38.

12. The multispecific antibody according to claim 2, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 21, 37 or 38, and the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 23, 39 or 40.

13. The multispecific antibody according to claim 12, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 37, and the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

39.

14. The multispecific antibody according to claim 3, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 23, 39 or 40, and the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 21, 37 or 38.

15. The multispecific antibody of claim 14, wherein the first antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 39, and the third antigen-binding module comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

37.

16. The multispecific antibody according to claim 1, wherein the first antigen-binding module and the third antigen-binding module are selected from any one of the following: (1) A first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 21 and a third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 23, 39 or 40; (2) A first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 37 and a third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 23, 39 or 40; (3) A first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 38 and a third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 23, 39 or 40; (4) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 21 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 23, 39 or 40; (5) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 37 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 23, 39 or 40; (6) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 38 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 23, 39 or 40; (7) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 41 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 42; or (8) A third antigen-binding module comprising the amino acid sequence shown in SEQ ID NO: 42 and a first antigen-binding module comprising the amino acid sequence shown in SEQ ID NO:

41.

17. The multispecific antibody according to claim 2, wherein the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:39, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:

37.

18. The multispecific antibody according to claim 3, wherein the third antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:37, and the first antigen-binding module comprises the amino acid sequence shown in SEQ ID NO:

39.

19. The multispecific antibody according to claim 1, wherein the single variable domain is of camel origin or humanized.

20. The multispecific antibody according to claim 1, wherein the second antigen-binding module is Fab, ScFv, or ScFab.

21. The multispecific antibody according to claim 1, wherein the second antigen-binding module comprises a heavy chain variable region having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 49 and a light chain variable region having at least 85% identity with the amino acid sequence shown in SEQ ID NO:

50.

22. The multispecific antibody according to claim 21, wherein the heavy chain variable region of the second antigen-binding module comprises the amino acid sequence shown in SEQ ID NO: 49, and its light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

50.

23. The multispecific antibody according to claim 1, wherein the amino acid sequence of the third antigen-binding module is as shown in SEQ ID NO: 37, the amino acid sequence of the first antigen-binding module is as shown in SEQ ID NO: 39; the amino acid sequence of the heavy chain variable region of the second antigen-binding module is as shown in SEQ ID NO: 49, and the amino acid sequence of its light chain variable region is as shown in SEQ ID NO:

50.

24. The multispecific antibody according to claim 1, wherein the second antigen-binding module is murine, chimeric, or humanized.

25. The multispecific antibody according to claim 1, wherein the third antigen-binding module and the first antigen-binding module are fused together.

26. The multispecific antibody according to claim 25, wherein the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module.

27. The multispecific antibody according to claim 1, wherein the third antigen-binding module and the second antigen-binding module are fused together.

28. The multispecific antibody of claim 27, wherein the third antigen-binding module is fused at its C-terminus to the N-terminus of the second antigen-binding module.

29. The multispecific antibody according to any one of claims 1-28, further comprising: (iv) The Fc domain consisting of two Fc polypeptides.

30. The multispecific antibody according to claim 29, wherein the second antigen-binding module is ScFv, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module.

31. The multispecific antibody according to claim 29, wherein the second antigen-binding module is ScFv, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the second antigen-binding module.

32. The multispecific antibody according to claim 29, wherein the second antigen-binding module is a Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of the first antigen-binding module.

33. The multispecific antibody according to claim 29, wherein the second antigen-binding module is a Fab, the first antigen-binding module is fused at its C-terminus to the N-terminus of one of the Fc peptides of the Fc domain, the second antigen-binding module is fused at its C-terminus to the N-terminus of another Fc peptide of the Fc domain, and the third antigen-binding module is fused at its C-terminus to the N-terminus of either the Fab light chain or the Fab heavy chain of the second antigen-binding module.

34. The multispecific antibody according to claim 29, wherein the Fc domain is an IgG Fc domain.

35. The multispecific antibody according to claim 34, wherein the IgG Fc domain is a human IgG Fc domain.

36. The multispecific antibody of claim 29, wherein the Fc domain comprises an amino acid substitution that promotes association between two Fc polypeptides of the Fc domain.

37. The multispecific antibody of claim 36, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions T366Y / W and S354C, and the other Fc polypeptide comprises amino acid substitutions Y407T / V, Y349C, T366S, and L368A.

38. The multispecific antibody of claim 29, wherein the Fc domain comprises amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function.

39. The multispecific antibody according to claim 38, wherein, according to the EU designation, the amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or the effector function are: both Fc polypeptides of the Fc domain contain amino acid substitutions L234A, L235A and P329G, or both contain amino acid substitutions L234A, L235A and P329A.

40. The multispecific antibody of claim 29, wherein the Fc domain comprises amino acid substitutions that reduce or eliminate the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A.

41. The multispecific antibody of claim 40, wherein, According to the EU designation, the Fc domain contains amino acid substitutions of H435R and / or Y436F that occur only in one of the Fc polypeptides.

42. The multispecific antibody according to claim 29, wherein, According to the EU designation, one of the Fc polypeptides in the Fc domain contains amino acid substitutions: L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide contains amino acid substitutions: L234A, L235A, P329A, S354C, T366W, H435R, and Y436F.

43. The multispecific antibody according to claim 1, wherein it is trivalent.

44. The multispecific antibody according to claim 1, wherein the multispecific antibody comprises: (1) Composed of two polypeptide chains, one of which contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 65, and the other polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

67. (2) Composed of two polypeptide chains, one of which contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 69, and the other polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

71. (3) Composed of three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 59, another polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 61, and a third polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 63; or, (4) Composed of three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 73, another polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO: 61, and a third polypeptide chain contains an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:

75.

45. The multispecific antibody according to claim 1, wherein the composition is: (1) It consists of two polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 65 and the other polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 67; (2) It consists of two polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 69 and the other polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 71; (3) Composed of three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 59, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 61, and the third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 63; or, (4) It consists of three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 73, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 61, and the third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

75.

46. ​​A multispecific antibody comprising two polypeptide chains, wherein the amino acid sequence of one polypeptide chain is shown in SEQ ID NO: 65 and the amino acid sequence of the other polypeptide chain is shown in SEQ ID NO:

67.

47. An isolated nucleic acid comprising a polynucleotide encoding a multispecific antibody according to any one of claims 1-46.

48. A vector comprising the nucleic acid according to claim 47.

49. A host cell comprising the nucleic acid according to claim 47, or the vector according to claim 48.

50. A method for preparing a multispecific antibody according to any one of claims 1-46, comprising culturing a host cell according to claim 49 to express the multispecific antibody, and isolating and purifying the multispecific antibody in the system.

51. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-46 and a pharmaceutically acceptable carrier.

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