Multispecific antibodies targeting BCMA

Abstract

Multispecific antibodies targeting BCMA are provided, in particular multispecific antibodies, nucleic acids encoding them, vectors comprising said nucleic acids, cells comprising said vectors, pharmaceutical compositions comprising them, and uses thereof for treating subjects with diseases associated with BCMA expression.

Description

[Technical field]

[0001] The present invention relates to multispecific antibodies, in particular multispecific antibodies that target 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 humans.

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

[0004] Multiple myeloma is a malignant plasma cell dyscrasia. In multiple myeloma cells, the expression level of BCMA is significantly increased. BCMA can be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma. Immunotherapeutic agents such as BCMA-binding antibodies can block the binding of BCMA to its natural ligands BAFF and / or APRIL.

[0005] In addition to the conventional four-chain antibody IgG1, which contains light and heavy chains, IgG antibodies containing only heavy chains (HcAb) IgG2 and IgG3 naturally exist in camelids. H H or single variable domains (VVs) have the characteristic of binding specifically to an antigen and have a high affinity for the antigen. H When used alone or as part of a larger antigen-binding molecule, the H domain has significant advantages over conventional scFv, antibody fragments, Fab, etc., such as the fact that only one domain is required for specific binding to an antigen with high affinity, that it can be converted into a multivalent or multispecific form, and that the V domain can be easily transformed into a multivalent or multispecific form. H The advantages of H domains include high solubility and no tendency to aggregate, high tissue permeability due to their small size, the fact that they are single-domain antibodies that do not require pairing with light chains, and the fact that there is no mispairing of light and heavy chains when forming multispecific antibodies.

[0006] Multispecific antibodies targeting B-cell maturation antigen (BCMA) and the T-cell CD3 receptor can redirect CD3-expressing T cells to BCMA-expressing myeloma cells, thereby inducing cytotoxicity against the target cells.

[0007] Antibodies targeting BCMA as a potential therapeutic target have been developed, but are still insufficient and more available options are needed. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides antibodies that target BCMA, in particular multispecific antibodies that target BCMA and a T cell activation antigen such as CD3. The present invention further provides nucleic acids, vectors, cells, compositions, methods of production and uses capable of encoding the provided antibodies. [Means for solving the problem]

[0009] In one aspect, the present invention provides a method for producing a composition comprising: (i) a first antigen-binding portion that binds to a first antigen; and (ii) a second antigen-binding portion that binds to a second antigen; and (iii) a third antigen-binding moiety that binds to the first antigen, the first antigen is BCMA and the second antigen is a T cell activation antigen, and the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains, each independently comprising: (a) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9; (b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or (d) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18 The present invention provides a multispecific antibody comprising any one of the following:

[0010] In some embodiments, the first antigen-binding portion and the third antigen-binding portion each independently comprise: (b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, or (c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15 Contains one of the following:

[0011] In some embodiments, the first antigen binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and the third antigen binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15. Further, in some embodiments, the first antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 41, and the third antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 42. In some embodiments, the first antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 37, and the third antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 39.

[0012] In another embodiment, the third antigen binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and the first antigen binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15. Further, in some embodiments, the first antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 42, and the third antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 41. In some embodiments, the first antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 39, and the third antigen binding portion comprises the amino acid sequence represented by SEQ ID NO: 37.

[0013] In some embodiments, the first antigen-binding moiety and the third antigen-binding moiety bind to different epitopes of BCMA.

[0014] The second antigen-binding portion confers the ability to bind to a T cell activation antigen. In some embodiments, the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising, as complementarity determining regions, HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 43, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, and the light chain variable region comprising LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 48. In a more specific embodiment, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 50.

[0015] In some embodiments, the multispecific antibody further comprises an Fc region comprised of two Fc polypeptides.

[0016] In some embodiments, in the multispecific antibody, the first antigen-binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; the third antigen-binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15; and the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises, as complementarity determining regions, an HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 43, an HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and an HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 45; and the light chain variable region comprises an LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 48. In a more specific embodiment, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 41, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 42, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 49 and the light chain variable region thereof comprises the amino acid sequence represented by SEQ ID NO: 50. In a more specific embodiment, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 37, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 39, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 49 and the light chain variable region thereof comprises the amino acid sequence represented by SEQ ID NO: 50. In these embodiments, in one alternative configuration, the first antigen-binding portion and the third antigen-binding portion are both single variable domains, the second antigen-binding portion is an ScFv, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the second antigen-binding portion.In a more specific embodiment, the multispecific antibody is composed of two polypeptide chains, of which a first polypeptide chain contains the above-mentioned first antigen-binding moiety and one Fc polypeptide in the Fc region, and a second polypeptide chain contains the above-mentioned second antigen-binding moiety, a third antigen-binding moiety and the other Fc polypeptide in the Fc region.

[0017] In another embodiment, in the multispecific antibody, the third antigen-binding portion comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; the first antigen-binding portion comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15; and the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises, as complementarity determining regions, HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 43, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 48. In a more specific embodiment, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 42, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 41, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 49 and the light chain variable region thereof comprises the amino acid sequence represented by SEQ ID NO: 50. In a more specific embodiment, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 39, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 37, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 49 and the light chain variable region thereof comprises the amino acid sequence represented by SEQ ID NO: 50. In these embodiments, in one alternative configuration, the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains, the second antigen-binding moiety is a Fab, the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is fused at the C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding moiety is fused at its C-terminus to the N-terminus of the first antigen-binding moiety.More specifically, for this embodiment, the multispecific antibody is composed of three polypeptide chains, of which the first polypeptide chain contains the first antigen-binding moiety, the third antigen-binding moiety and one Fc polypeptide in the Fc region, the second polypeptide chain contains the Fab heavy chain of the second antigen-binding moiety and the other Fc polypeptide in the Fc region, and the third polypeptide chain is the Fab light chain of the second antigen-binding moiety. In another alternative configuration, the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains, the second antigen-binding moiety is an ScFv, and the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding moiety is fused at its C-terminus to the N-terminus of the first antigen-binding moiety. More specifically, for this embodiment, the multispecific antibody is composed of two polypeptide chains, of which the first polypeptide chain contains the first antigen-binding moiety, the third antigen-binding moiety and one Fc polypeptide in the Fc region, and the second polypeptide chain contains the second antigen-binding moiety and the other Fc polypeptide in the Fc region. In another alternative configuration, the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains, the second antigen-binding moiety is a Fab, and the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is fused at its C-terminus of the Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding moiety is fused at its C-terminus to the N-terminus of the Fab light chain of the second antigen-binding moiety. More specifically for this embodiment, the multispecific antibody is composed of three polypeptide chains, of which a first polypeptide chain contains the first antigen-binding moiety and one Fc polypeptide in the Fc region, a second polypeptide chain contains a Fab heavy chain of the second antigen-binding moiety and the other Fc polypeptide in the Fc region, and a third polypeptide chain contains the third antigen-binding moiety and the Fab light chain of the second antigen-binding moiety.

[0018] In one aspect, the invention provides an isolated nucleic acid comprising a polynucleotide encoding the multispecific antibody described above.

[0019] In one aspect, the invention provides a vector comprising a nucleic acid according to the invention.

[0020] In one aspect, the invention provides a host cell comprising a nucleic acid or vector according to the invention.

[0021] In another aspect, the present invention provides a method for producing the multispecific antibody according to the present invention, comprising culturing the host cell described above to express the multispecific antibody, and isolating and purifying the multispecific antibody from the system.

[0022] In another aspect, the present invention provides a pharmaceutical composition comprising the multispecific antibody described above and a pharma- ceutically acceptable carrier.

[0023] In another aspect, the present invention provides the use of said multispecific antibody or said pharmaceutical composition for the manufacture of a medicament for the treatment of a disease associated with BCMA expression.

[0024] In another aspect, the present invention provides a method for treating a disease associated with BCMA expression in a subject, comprising administering to the subject a therapeutically effective amount of said multispecific antibody or said pharmaceutical composition.

[0025] In another aspect, the present invention provides a method for producing a method for manufacturing a pharmaceutical composition comprising: (1) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9; (2) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (3) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or (4) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18; The present invention provides a BCMA binding antibody comprising a single variable domain comprising:

[0026] In some embodiments, the single variable domain comprises the amino acid sequence represented by SEQ ID NO:41 or 42, but does not comprise the amino acid sequence represented by SEQ ID NO:21 or 23.

[0027] The present invention provides multispecific antibodies that target BCMA and T cell activation antigens such as CD3. In some forms, various forms of multispecific antibodies have been constructed using specific single variable domains, and all of them have shown good anti-tumor effects, such as tumor cell lysis and safety. [Brief description of the drawings]

[0028] [Figure 1A] FIG. 1A is a flow cytometric binding curve of anti-human BCMA VHH-Fc chimeric antibody to CHO-hBCMA cells. [Figure 1B] FIG. 1B is a binding curve of anti-human BCMA VHH-Fc chimeric antibody to U266 cells by flow cytometry. [Figure 1C] FIG. 1C is a binding curve of anti-human BCMA VHH-Fc chimeric antibody to RPMI8226 cells by flow cytometry. [Figure 1D] FIG. 1D is a binding curve of anti-human BCMA VHH-Fc chimeric antibody to HUVEC cells by flow cytometry. [Diagram 2] Figure 2 shows binding curves of 1A10-Fc and 1A11-Fc chimeric antibodies to HEK293T-CynoBCMA or HEK293T measured by flow cytometry. [Diagram 3] FIG. 3 shows the results of competitive ELISA measurements of anti-human BCMA VHH-Fc chimeric antibody and the ligand APRIL. [Figure 4A] Figure 4A shows a comparison of the amino acid sequences in the extracellular domains of human and cynomolgus monkey BCMA. [Figure 4B] FIG. 4B shows the crystal structure of the extracellular domain of human BCMA. [Diagram 5] FIG. 5 is a schematic diagram showing the structure of one example of a multispecific antibody according to the present invention. [Figure 6] FIG. 6 is a schematic diagram showing the structures of exemplary antibodies BC24, PC1, PC2 and PC3. [Figure 7] FIG. 7 shows the binding curve of anti-BCMA / anti-CD3 multispecific antibody to NCI-H929 cells. [Figure 8] FIG. 8 shows the binding curve between anti-BCMA / anti-CD3 multispecific antibodies and MM1S cells. [Figure 9] FIG. 9 shows the binding curve of anti-BCMA / anti-CD3 multispecific antibodies to RPMI-8226 cells. [Figure 10] FIG. 10 shows the binding curve between anti-BCMA / anti-CD3 multispecific antibodies and CD3+ T cells. [Figure 11] FIG. 11 shows the rate of lysis of NCI-H929 tumor cells by effector cells induced by anti-BCMA / anti-CD3 multispecific antibodies at each concentration. [Figure 12] FIG. 12 shows the rate of lysis of MM1S tumor cells by effector cells induced by anti-BCMA / anti-CD3 multispecific antibodies at each concentration. [Figure 13] FIG. 13 shows the rate of RPMI-8226 tumor cell lysis by effector cells induced by anti-BCMA / anti-CD3 multispecific antibodies at each concentration. [Figure 14]FIG. 14 shows the effect of anti-BCMA / anti-CD3 multispecific antibodies on T cell activation in the presence of target cells NCI-H929. [Figure 15] FIG. 15 shows the effect of anti-BCMA / anti-CD3 multispecific antibodies on T cell activation in the presence of target cells MM1S. [Figure 16] FIG. 16 shows the release of cytokines IL-2, IL-6, TNF-α, and IFN-γ after 24 hours of incubation of anti-BCMA / anti-CD3 antibodies with NCI-H929 cells and effector cells at various concentrations. [Figure 17] FIG. 17 shows the effect of anti-BCMA / anti-CD3 antibodies on the growth of NCI-H929 subcutaneously implanted tumors. [Figure 18] FIG. 18 shows the results of LC-MS analysis of anti-BCMA / anti-CD3 antibody PC1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] [term] The term "antibody" refers to a protein that contains an antigen-binding site, and includes natural antibodies and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, single-specific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), and single-chain antibodies.

[0030] The term "multispecific" means that an antibody can specifically bind to multiple different antigenic determinants, e.g., can specifically bind to two or more different antigenic determinants. Typically, a bispecific antibody contains two antigen binding sites, each specific for a different antigenic determinant. The different antigenic determinants can be expressed on the same cell or on different cells. The different antigenic determinants can be different depending on the type of antigen (e.g., those that bind to antigen CD3, BCMA) or can be present on the same antigen. An antigenic determinant is a special chemical group with a specific composition and structure on the surface or other part of an antigenic molecule, which is a structure that can specifically bind to a corresponding antibody or sensitized lymphocyte. As an example of an antigenic determinant, the antigenic substance B cell maturation antigen (BCMA) has various antigenic determinants with confirmed or unknown structure. In this specification, an antibody that can bind to two or more different antigenic determinants on an antigen is called a multispecific antibody.

[0031] The term "N-valent antibody" means that an antibody has N antigen-binding sites. For example, a "bivalent antibody" means that an antibody has two antigen-binding sites, and a "trivalent antibody" means that an antibody has three antigen-binding sites. A naturally occurring human immunoglobulin molecule typically has two antigen-binding sites. A Fab typically has one antigen-binding site. A single variable domain, ScFv, typically has one antigen-binding site.

[0032] The term "antigen-binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. Particular antigen-binding portions may be Fab, ScFv or single variable domains. Synonymous herein with antigenic determinant and epitope.

[0033] The term "T cell activation antigen" refers to an antigen expressed on the surface of a T lymphocyte, such as a cytotoxic T lymphocyte, that can induce T cell activation upon interaction with an antibody. For example, interaction of an antibody with a T cell activation antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In one embodiment, the T cell activation antigen is CD3, e.g., the epsilon chain of CD3.

[0034] 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. The multispecific antibodies of the present invention are capable of inducing T cell activation. Suitable methods for measuring T cell activation are known in the art and described herein.

[0035] In the present invention, the terms "first", "second" or "third" in reference to antigen-binding moieties, antigens, Fc polypeptides, peptide linkers, polypeptide chains, etc. are used for convenience of distinguishing when more than one of each type of moiety is present. The use of these terms is not intended to confer a particular order or orientation of the multispecific antibodies, unless expressly stated.

[0036] The term "fused" means that components (eg, Fab, ScFv, Fc polypeptides, etc.) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0037] The term "variable domain" or "variable region" refers to the domain of an antibody that is involved in binding of the antibody to an antigen. For example, naturally occurring four-chain antibodies (e.g., from humans, murines, etc.) have a heavy chain variable region (VH) and a light chain variable region (VL), while heavy chain-only antibodies from animals such as camelids and sharks have a single variable domain. Each variable domain of a naturally occurring antibody is generally composed of four "framework regions" and three "complementarity determining regions". The four framework regions are referred to as framework region 1 (or FR1), framework region 2 (or FR2), framework region 3 (or FR3), and framework region 4 (or FR4), respectively. The framework regions are separated by three complementarity determining regions (or CDRs), which are referred to in the art and hereinafter as complementarity determining region 1 (or CDR1), complementarity determining region 2 (or CDR2), and complementarity determining region 3 (or CDR3), respectively. Thus, the general structure of a variable domain can be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain contains the antigen-binding site and thus confers specificity for the antigen to the antibody.

[0038] The term "single variable domain" refers to a variable domain that is capable of specifically binding to an epitope without pairing with another variable domain. A single variable domain typically has three CDRs (CDR1, CDR2 and CDR3) present in one domain. In some cases, the single variable domain may be a heavy chain variable domain (e.g., VH), provided that it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain) (in which case the single antigen-binding domain is capable of forming a functional antigen-binding unit without interacting with another variable domain). Another example of a single variable domain is the camelid "VHH domain" (or "VHH" or "VHH"). H It is abbreviated as "H".

[0039] "CDR" (complementarity determining region), also called "hypervariable region (HVR)", generally refers to each region of an antibody variable region that frequently varies in sequence and / or forms structurally defined loops. Naturally occurring four-chain antibodies generally 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 domains generally have three CDRs (CDR1, CDR2 and CDR3).

[0040] Several methods for defining and classifying CDRs have been reported. Among them, the Kabat definition, which classifies CDRs based on sequence variability, is the most widely used (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md .(1991)). 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 in Oxford Molecular's AbM antibody structure modeling software. The "contact" definition of CDRs is based on the analysis of available complex crystal structures. However, the boundaries of CDRs of the same antibody variable region may differ depending on the definition and classification method. That is, the CDR sequences of the same antibody variable region classified and defined by different methods may differ. Therefore, when an antibody is defined using a specific CDR sequence classified and defined in the present invention, the scope of the antibody also includes antibodies defined by CDR sequences converted to any other definition (e.g., Chothia, AbM, etc.).

[0041] The CDR sequences described herein generally follow the Kabat definition (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0042] The term "framework region" or "FR" refers to amino acid residues of a variable domain other than the CDR residues as herein defined.

[0043] The term "Fab" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain and the VL and CL domains of a light chain. As used herein, Fab refers to a native Fab molecule that includes a Fab heavy chain (VH-CH1, N-to-C-terminal direction) consisting of a heavy chain variable region VH and a constant region CH1, and a Fab light chain (VL-CL, N-to-C-terminal direction) consisting of a light chain variable region and a constant region CL.

[0044] The term "scFv" comprises the VH and VL domains of an immunoglobulin, wherein these domains are present in a single polypeptide chain. In some embodiments, the scFv also comprises a peptide linker between the VH and VL domains such that it can form the necessary structure for antigen binding.

[0045] The term "Fc domain", "Fc" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc and variant Fc. The C-terminal lysine (Lys447) of Fc may or may not be present. Unless otherwise specified, the numbering of amino acid residues in an Fc or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. As used herein, a "Fc polypeptide" in an Fc region refers to one of the two polypeptides that form a dimeric Fc domain. For example, the Fc polypeptide of an IgG Fc region includes the IgG CH2 and IgG CH3 constant regions.

[0046] The term "effector function" refers to the biological activities attributable to the antibody Fc domain, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), etc.

[0047] The term "KD" as used herein refers to dissociation constant and is expressed as molar concentration (M). The KD value of an antibody can be measured by methods known in the art. One example of a method for measuring the KD of an antibody is a method using surface plasmon resonance, for example, a biosensor system such as a Biacore system.

[0048] The term "treatment" refers to a clinical intervention that seeks to alter the natural course of a disease in an individual undergoing treatment, and can be performed prophylactically or during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or reduction of the disease state, and improvement of remission or prognosis.

[0049] The term "subject" includes any human or non-human animal. "Non-human animals" encompass all vertebrates, including mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. Preferably, the subject of the present invention is a human. Unless otherwise specified, "patient" or "subject" are used interchangeably.

[0050] The term "isolated" means that the compound of interest (eg, a VHH, a multispecific antibody, an antibody or a nucleic acid, etc.) has been removed from its natural environment.

[0051] "Percentage identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence shown herein after aligning the candidate sequence with the specific amino acid sequence shown herein and introducing gaps as necessary to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. Amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared.

[0052] Various aspects of the invention are described in further detail in the following subsections.

[0053] I. Single Variable Domains The present invention provides single variable domains that bind to BCMA (e.g., human BCMA). Single variable domains provide further options available for drug development or drug construction targeting BCMA. The single variable domains can provide targeting properties with good affinity to human BCMA. In some cases, the single variable domains can provide the ability to block the binding of the proliferation-inducing ligand APRIL to BCMA. Notably, in some forms, the single variable domains do not bind to human TACI and BAFF-R proteins, demonstrating specificity. In some embodiments, the single variable domains cross-react with monkey BCMA. Cross-reactivity with monkeys is favorable for developing drug toxicity experiments, since monkeys are ideal experimental animals for drug toxicity experiments.

[0054] The present invention relates to (1) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9; (2) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (3) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or (4) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18; The present invention provides a single variable domain that binds to BCMA, comprising:

[0055] In some embodiments, the single variable domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the single variable domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the single variable domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the single variable domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the single variable domain is camelid. In some embodiments, the single variable domain is humanized.

[0056] 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 to the sequence represented by 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 to the sequence represented by SEQ ID NO: 19, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence represented by 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 to the sequence set forth in SEQ ID NO: 41, and comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth 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 to the sequence set forth in SEQ ID NO: 37 or 38, and comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth 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 to the sequence set forth in SEQ ID NO:42, and comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and a CDR3 comprising the amino acid sequence set forth 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 to the sequence represented by SEQ ID NO: 39 or 40, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by 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 to the sequence represented by SEQ ID NO: 25, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18. In some embodiments, an 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 compared to a reference sequence, but a single variable domain comprising the sequence retains the ability to bind BCMA. In some embodiments, an amino acid sequence selected from SEQ ID NOs: 19, 41, 42 and 25 has a total of 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 amino acids substituted, inserted and / or deleted. In some embodiments, the substitutions, insertions or deletions occur in regions other than the CDRs (i.e. the FRs). In some embodiments, the substitution, insertion or deletion occurs in one, two or three of the CDR regions, e.g., CDR1, CDR2, CDR3. In some embodiments, the substitution, insertion or deletion occurs in the CDR regions and in non-CDR regions.

[0057] In some embodiments, the amino acid sequence of the single variable domain comprises the amino acid sequence represented by SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, 40, 41 or 42.

[0058] In some embodiments, the amino acid sequence of the single variable domain is represented by SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, 40, 41 or 42.

[0059] In some embodiments, the single variable domain is a V H H. In some embodiments, V H H is humanized. Single variable domains of non-human origin can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the original single variable domain sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a human antibody. Humanization is expected to reduce immunogenicity. Single variable domains generally have a structure represented from the N-terminus to the C-terminus as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0060] In some embodiments, the invention provides a single variable domain that binds to the same epitope as any of the single variable domains according to the invention. In some embodiments, the invention provides a single variable domain that binds to the same epitope as a single variable domain comprising an amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 25, 37, 38, 39 or 40. In some embodiments, the single variable domain that binds to the same epitope is camelid or humanized.

[0061] Single variable domains can be screened for competitive binding to the same epitope using conventional techniques known to those skilled in the art. Thus, in some embodiments, the invention provides single variable domains that bind to BCMA in competition with any of the single variable domains according to the invention. In some embodiments, the invention provides single variable domains that bind to BCMA in competition with a single variable domain comprising an amino acid sequence represented by SEQ ID NO: 19, 21, 23, 25, 37, 38, 39 or 40. Binding to BCMA can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) or any other method known in the art. In some embodiments, the single variable domain that competitively binds to BCMA is camelid or humanized.

[0062] The present invention provides several examples of single variable domains that bind BCMA. The amino acid sequences of the CDRs (CDR1, CDR2 and CDR3) of example single variable domains according to the invention are shown in Table S1 below. The full length amino acid sequences of example single variable domains are shown in Table S2 below.

[0063] [Table 1]

[0064] [Table 2]

[0065] (Sequence of SEQ ID NO: 41) QVQLVESGGGX1VQX2GGSLRLSCAASGYSSNVACMAWYRQAPGKX3X4EWVX5TIVADFGTTNYAASVKGRFTISQDNX6KNTVYLQMNSLX7X8EDX9AX 10 YYCAATQRGGIDWCDEINYWGQGTLVTVSS 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, and X 10 is V or M.

[0066] (Sequence of SEQ ID NO: 42) QVQLVESGGGX 11 VQX 12 GGSLRLSCAASGX 13 TFNSACMGWFRQAPGKX 14 REGVX 15 RIETGYGGTVYADSVKGRFTISRDNX 16 KNTVYLQMNSLX 17 X 18 EDTAX 19 YYCAAKRSWCTPTWWHELDYNYWGQGTQVTVSS Where X 11 is S or L, and X 12 is P or A, and X 13 is F or V, and X 14 is E or G, and X 15 is A or S, and X 16 is A or S, and X 17 is R or K, and X 18 is A or P, and X 19 is V or M.

[0067] The present invention provides a BCMA-binding antibody comprising the above-described single variable domain.

[0068] In some embodiments, the BCMA binding antibody may be a monospecific antibody or a multispecific antibody.

[0069] In one embodiment, the monospecific antibody comprises an Fc domain, preferably wherein the Fc is a human IgG1, IgG2, IgG3 or IgG4 Fc.

[0070] 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 to the sequence set forth in SEQ ID NO: 27, 29, 31, 33, 51, 53, 55 or 57.

[0071] The present invention provides examples of monospecific antibodies (e.g., 1A10-V1, 1A10-V1, 1A11-V1, 1A11-V2, etc.) in which the single variable domains are fused to the Fc of human IgG1 and form homodimers with the Fc.

[0072] The invention provides an isolated nucleic acid comprising a polynucleotide encoding a BCMA binding antibody according to the invention.

[0073] The present invention provides a vector comprising a nucleic acid according to the present invention.

[0074] The present invention provides an isolated host cell comprising a nucleic acid according to the invention or a vector according to the invention.

[0075] The present invention provides a method for producing a BCMA-binding antibody according to the present invention, comprising culturing a host cell according to the present invention to express the BCMA-binding antibody, and isolating and purifying the BCMA-binding antibody from the system.

[0076] The invention provides a pharmaceutical composition comprising a BCMA binding antibody according to the invention and a pharma- ceutically acceptable carrier.

[0077] The invention provides a method for treating a disease associated with BCMA expression in a subject comprising administering to the subject a therapeutically effective amount of a BCMA binding antibody according to the invention or said pharmaceutical composition.

[0078] II. Multispecific antibodies The present invention provides multispecific antibodies comprising single variable domains that are capable of targeting BCMA expressed on the surface of tumor cells and are also capable of binding to T cells and activating the tumoricidal activity of the T cells.

[0079] Mispairing of light and heavy chains can be reduced or avoided when a single variable domain is employed compared to when more (eg, two) Fabs are employed.

[0080] The multispecific antibodies of the present invention also exhibit excellent antitumor properties, such as tumor cell lysis. In some embodiments, the multispecific antibodies of the present invention can induce killing of various tumor cells, and the multispecific antibodies of the present invention exhibit good tumoricidal activity against tumor cells with different BCMA expression levels. In particular, they exhibit better killing properties against some tumor cells with low BCMA expression.

[0081] The multispecific antibodies of the present invention exhibit good antitumor properties and, due to their low levels of cytokine release, can reduce the risk of cytokine storm caused by CD3-targeting antibodies and improve safety.

[0082] Multispecific antibodies can be constructed using any of the single variable domains described in section I above. Thus, the present invention provides (i) a first antigen-binding portion that binds to a first antigen; and (ii) a second antigen-binding portion that binds to a second antigen; and (iii) a third antigen-binding moiety that binds to the first antigen, the first antigen is BCMA and the second antigen is a T cell activation antigen, and the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains, each independently comprising: (a) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9; (b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or (d) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18 Contains one of the following: Multispecific antibodies are provided.

[0083] In the present invention, the first and third antigen-binding moieties are both single variable domains that bind to BCMA. In some embodiments, the first antigen-binding moiety comprises a complementarity determining region as described in (a), (b), (c) or (d) above. In some embodiments, the third antigen-binding moiety comprises a complementarity determining region as described in (a), (b), (c) or (d) above. In the multispecific antibody, the first and third antigen-binding moieties may be the same or different, and may be independently combined with any single variable domain according to the present invention. In one embodiment, the first and third antigen-binding moieties are the same. In another embodiment, the first and third antigen-binding moieties are different.

[0084] Table S3 illustrates multispecific antibodies constructed with various combinations of a first antigen-binding moiety characterized by CDRs and a third antigen-binding moiety. In one embodiment, the first antigen-binding moiety comprises the complementarity determining region described in (b) above, and the third antigen-binding moiety comprises the complementarity determining region described in (c) above, i.e., the first antigen-binding moiety comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and the third antigen-binding moiety comprises CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15. In another embodiment, the third antigen-binding portion comprises the complementarity determining region described in (b) above, and the first antigen-binding portion comprises the complementarity determining region described in (c) above, i.e., the third antigen-binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and the first antigen-binding portion comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15. In a more specific embodiment, the first antigen-binding portion comprises a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12, and the third antigen-binding portion comprises a CDR1 represented by SEQ ID NO: 13, a CDR2 represented by SEQ ID NO: 14, and a CDR3 represented by SEQ ID NO: 15. In another more specific embodiment, the third antigen-binding portion comprises a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12, and the first antigen-binding portion comprises a CDR1 represented by SEQ ID NO: 13, a CDR2 represented by SEQ ID NO: 14, and a CDR3 represented by SEQ ID NO: 15.

[0085] [Table 3]

[0086] (Note) "(X)+(Y)" in the table indicates the form of a combination of the third antigen-binding portion and the first antigen-binding portion in a multispecific antibody. For example, (b)+(a) indicates that the third antigen-binding portion in the multispecific antibody contains the complementarity determining region described in (b) and the first antigen-binding portion contains the complementarity determining region described in (a).

[0087] Further, in some embodiments, the first antigen-binding portion and the third antigen-binding portion 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 to a sequence set forth in SEQ ID NO: 19, 41, 42 or 25.

[0088] In some embodiments, the first antigen binding portion 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 to a sequence represented by SEQ ID NO: 19, 41, 42 or 25. In some embodiments, the first antigen binding portion 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 to a sequence represented by SEQ ID NO: 19, 21, 23, 37, 38, 39, 40 or 25. In some embodiments, the first antigen binding portion comprises an amino acid sequence represented by SEQ ID NO: 19, 41, 42 or 25. In some more specific embodiments, the first antigen-binding portion comprises an amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 37, 38, 39, 40, or 25.

[0089] In some embodiments, the third antigen binding portion 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 to the sequence represented by SEQ ID NO: 19, 41, 42 or 25. In some embodiments, the third antigen binding portion 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 to the sequence represented by SEQ ID NO: 19, 21, 23, 37, 38, 39, 40 or 25. In some embodiments, the third antigen binding portion comprises an amino acid sequence represented by SEQ ID NO: 19, 41, 42 or 25. In some more specific embodiments, the third antigen-binding portion comprises an amino acid sequence set forth in SEQ ID NO: 19, 21, 23, 37, 38, 39, 40, or 25.

[0090] In some embodiments, the first antigen binding portion 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 to the sequence set forth in SEQ ID NO: 41, and the third antigen binding portion 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 to the sequence set forth in SEQ ID NO: 42. In one form, the first antigen binding portion comprises an amino acid sequence set forth in SEQ ID NO: 41, and the third antigen binding portion comprises an amino acid sequence set forth in SEQ ID NO: 42. In some more specific embodiments, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:21, 37, or 38, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:23, 39, or 40.

[0091] In some embodiments, the first antigen binding portion 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 to the sequence set forth in SEQ ID NO: 42, and the third antigen binding portion 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 to the sequence set forth in SEQ ID NO: 41. In one form, the first antigen binding portion comprises an amino acid sequence set forth in SEQ ID NO: 42, and the third antigen binding portion comprises an amino acid sequence set forth in SEQ ID NO: 41. In some more specific embodiments, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:23, 39, or 40, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:21, 37, or 38.

[0092] In one example, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:21, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:21, 23, 37, 38, 39, or 40.

[0093] In one example, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:21, and the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:21, 23, 37, 38, 39, or 40.

[0094] In one example, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:37, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:39.

[0095] In one example, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:37, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:38.

[0096] In one example, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:37, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:40.

[0097] In one example, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:38, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:39.

[0098] In one example, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:38, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:40.

[0099] In one example, the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:39, and the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:40.

[0100] In one example, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:37, and the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:39.

[0101] In one example, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:37, and the first antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:38.

[0102] In one example, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:37, and the first antigen-binding portion comprises the amino acid sequence of SEQ ID NO:40.

[0103] In one example, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:38, and the first antigen-binding portion comprises the amino acid sequence of SEQ ID NO:39.

[0104] In one example, the third antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO:38, and the first antigen-binding portion comprises the amino acid sequence of SEQ ID NO:40.

[0105] In one example, the third antigen-binding portion comprises the amino acid sequence set forth in SEQ ID NO:39, and the first antigen-binding portion comprises the amino acid sequence of SEQ ID NO:40.

[0106] In one example, the first antigen-binding portion and the third antigen-binding portion both comprise the amino acid sequence set forth in SEQ ID NO: 21, 23, 37, 38, 39 or 40.

[0107] Table S4 illustrates multispecific antibodies constructed with various combinations of first and third antigen-binding moieties characterized by full-length amino acid sequences.

[0108] [Table 4]

[0109] (Note) "(X)+(Y)" in the table represents the combination of the third antigen-binding moiety and the first antigen-binding moiety in the multispecific antibody. For example, "1A10+1A1 single variable domain" represents that the third antigen-binding moiety in the multispecific antibody is the amino acid sequence of the 1A10 single variable domain, and the first antigen-binding moiety is the amino acid sequence of the 1A1 single variable domain. Please refer to Table S2 for the amino acid sequences of the single variable domains in the above table.

[0110] In the present invention, the first antigen-binding moiety may be from a camelid or may be humanized. The third antigen-binding moiety may be from a camelid or may be humanized. Humanization can reduce immunogenicity. In some embodiments, both the first and third antigen-binding moieties are humanized. In some embodiments, both the first and third antigen-binding moieties are from a camelid.

[0111] In the multispecific antibody of the invention, the second antigen-binding moiety provides the ability to target a T cell activation antigen. The second antigen-binding moiety may be a Fab, ScFv or ScFab. In one embodiment, the T cell activation antigen is CD3. In one form, the second antigen-binding moiety is a CD3-binding Fab. In another form, the second antigen-binding moiety is a CD3-binding ScFv.

[0112] In some embodiments, the second antigen-binding portion is murine, chimeric, or humanized.

[0113] In some embodiments, the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising, as complementarity determining regions, an HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 43, an HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and an HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, and the light chain variable region comprising an LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 48.

[0114] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the variable region represented by SEQ ID NO:49, and the light chain variable region comprises LCDR1, HCDR2 and LCDR3 of the variable region represented by SEQ ID NO:50.

[0115] In some embodiments, the second antigen-binding portion comprises a heavy chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 49, and a light chain variable region comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO: 50. In one embodiment, the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence set forth in SEQ ID NO: 49, and the light chain variable region comprises the amino acid sequence set forth 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 portion are represented by SEQ ID NOs: 49 and 50, respectively, in that order.

[0116] [Table 5]

[0117] The multispecific antibody of the present invention may not contain an Fc region, and the first antigen-binding portion, the second antigen-binding portion and the third antigen-binding portion may be linked by a suitable linker.

[0118] The multispecific antibodies of the invention may comprise an Fc region, which can provide functions such as extended half-life and Fc region-associated effector functions.

[0119] Construction of multispecific antibodies The components of a multispecific antibody of the invention can be fused to each other in a variety of configurations, hi some embodiments, the multispecific antibody further comprises (iv) an Fc region composed of two Fc polypeptides.

[0120] The third antigen-binding moiety can be fused to the first antigen-binding moiety or the second antigen-binding moiety. In an alternative embodiment of pattern (1), the third antigen-binding moiety and the first antigen-binding moiety are fused to each other, optionally fused to each other via a peptide linker. In an alternative embodiment of pattern (2), the third antigen-binding moiety and the second antigen-binding moiety are fused to each other, optionally fused to each other via a peptide linker.

[0121] In an alternative embodiment of pattern (1), the third antigen-binding moiety and the first antigen-binding moiety are fused to each other, optionally fused to each other via a peptide linker. In a more specific embodiment, the third antigen-binding moiety is fused at its C-terminus to the N-terminus of the first antigen-binding moiety. Furthermore, in some embodiments, the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is an ScFv, and the second antigen-binding moiety is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region. In another embodiment, the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is a Fab, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region.

[0122] In one embodiment, both the first and third antigen-binding moieties are single variable domains, the second antigen-binding moiety is a Fab, the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is fused at its C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding moiety is fused at its C-terminus to the N-terminus of the first antigen-binding moiety. Such a structure is shown diagrammatically in FIG. 5A. More specifically for this embodiment, the multispecific antibody has three polypeptide chains, of which the first polypeptide chain contains the first antigen-binding moiety, the third antigen-binding moiety and one Fc polypeptide in the Fc region, the second polypeptide chain contains the Fab heavy chain of the second antigen-binding moiety and the other Fc polypeptide in the Fc region, and the third polypeptide chain is the Fab light chain of the second antigen-binding moiety.

[0123] In another embodiment, both the first and third antigen-binding moieties are single variable domains, the second antigen-binding moiety is an ScFv, and the first antigen-binding moiety is fused C-terminally to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is fused C-terminally to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding moiety is fused C-terminally to the N-terminus of the first antigen-binding moiety. Such a structure is shown diagrammatically in FIG. 5B. More specifically for this embodiment, the multispecific antibody has two polypeptide chains, of which the first polypeptide chain contains the first antigen-binding moiety, the third antigen-binding moiety and one Fc polypeptide in the Fc region, and the second polypeptide chain contains the second antigen-binding moiety and the other Fc polypeptide in the Fc region.

[0124] Regarding the linking form of the third antigen-binding portion, in an alternative embodiment of pattern (2), the third antigen-binding portion and the second antigen-binding portion are fused to each other, optionally fused to each other via a peptide linker. In a more specific embodiment, the third antigen-binding portion is fused at its C-terminus to the N-terminus of the second antigen-binding portion. Furthermore, in some embodiments, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is an ScFv, and the second antigen-binding portion is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region. In another embodiment, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is a Fab, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region. wherein the third antigen-binding portion is fused to the N-terminus of the Fab light chain or Fab heavy chain of the second antigen-binding portion.

[0125] In one embodiment, both the first and third antigen-binding moieties are single variable domains, the second antigen-binding moiety is an ScFv, the first antigen-binding moiety is fused C-terminally to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is fused C-terminally to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding moiety is fused C-terminally to the N-terminus of the second antigen-binding moiety. Such a structure is shown diagrammatically in FIG. 5C. More specifically for this embodiment, the multispecific antibody has two polypeptide chains, of which the first polypeptide chain contains the first antigen-binding moiety and one Fc polypeptide in the Fc region, and the second polypeptide chain contains the second antigen-binding moiety, the third antigen-binding moiety, and the other Fc polypeptide in the Fc region.

[0126] In another embodiment, both the first and third antigen-binding moieties are single variable domains, the second antigen-binding moiety is a Fab, the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding moiety is fused at its C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding moiety is fused at its C-terminus to the N-terminus of a Fab light chain of the second antigen-binding moiety (such a structure is shown diagrammatically in FIG. 5D ) or the N-terminus of a Fab heavy chain of the second antigen-binding moiety. More specifically for this embodiment, the multispecific antibody has three polypeptide chains, of which a first polypeptide chain contains the first antigen-binding moiety and one Fc polypeptide in the Fc region, a second polypeptide chain contains the Fab heavy chain of the second antigen-binding moiety and the other Fc polypeptide in the Fc region, and a third polypeptide chain contains the third antigen-binding moiety and the Fab light chain of the second antigen-binding moiety. Optionally, the multispecific antibody has three polypeptide chains, of which a first polypeptide chain contains the first antigen-binding moiety and one Fc polypeptide in the Fc region, a second polypeptide chain contains the Fab heavy chain of the second antigen-binding moiety, the third antigen-binding moiety and the other Fc polypeptide in the Fc region, and a third polypeptide chain contains the Fab light chain of the second antigen-binding moiety.

[0127] When the second antigen-binding portion is an ScFv, the heavy chain variable region (VH) and light chain variable region (VL) contained therein may be arranged in any order, but for example, in one embodiment, the VH and VL of the second antigen-binding portion are arranged in the order of VL-VH from the N-terminus to the C-terminus. In another embodiment, the VH and VL of the second antigen-binding portion are arranged in the order of VH-VL from the N-terminus to the C-terminus. In some embodiments, the VH and VL of the second antigen-binding portion are fused via a peptide linker to form an ScFv.

[0128] In any of the above embodiments, a person skilled in the art can appropriately select a feasible linkage between the components of the multispecific antibody, for example, a direct linkage, or a linkage via various peptide linkers (e.g., peptide linkers comprising one or more amino acids, typically about 1 to 50 amino acids) described herein or known in the art, or a hinge fusion.

[0129] The third antigen-binding moiety can be fused to the first or second antigen-binding moiety directly or via a peptide linker. In one embodiment, the third antigen-binding moiety is fused to the first or second antigen-binding moiety via a peptide linker. In some embodiments, when the second antigen-binding moiety is an ScFv structure, its light chain variable region and heavy chain variable region are linked via a peptide linker.

[0130] Each peptide linker may independently be any suitable one, such as, for example, a charged and / or flexible linker polypeptide. In a specific embodiment, the peptide linker is composed of 1 to 50 amino acids linked by peptide bonds, the amino acids being selected from the 20 natural 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, examples of peptide linkers include polyglycine (particularly (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.

[0131] In some embodiments, the peptide linker may be a peptide linker consisting 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 20 or more. In some embodiments, the peptide linker is a peptide linker containing repeated GGGGS, a peptide linker containing GGSGGSGGSGGSGG (SEQ ID NO: 2), or a peptide linker containing GGGPGKR. Preferably, the peptide linker containing repeated GGGGS is (GGGGS)n. Here, n is any number from 1 to 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or any range limited by any two of the numbers, e.g., 1 to 5, 2 to 5, 3 to 6, 2 to 4, 1 to 4, etc. In some embodiments, the peptide linker is a linker polypeptide comprising GGGGS, (GGGGS)2, (GGGGS)3, or (GGGGS)4. In some embodiments, the third antigen-binding moiety is fused to the first antigen-binding moiety or the second antigen-binding moiety via the peptide linker GGGGS (SEQ ID NO: 1) or (GGGGS)2 or (GGGGS)3. In some embodiments, when the second antigen-binding moiety is an ScFv structure, its light chain variable region and heavy chain variable region are linked via the peptide linker GGGGS or (GGGGS)2 or (GGGGS)3.

[0132] When fused to Fc, it is typically fused via the hinge region. In one embodiment, the first antigen-binding portion is fused to one of the Fc polypeptides in the Fc region via a first hinge, and the second antigen-binding portion is fused to one of the Fc polypeptides in the Fc region via a second hinge. In some embodiments, the first hinge and the second hinge can form a covalent bond, such as a disulfide bond, with each other. The first hinge or / and the second hinge may comprise amino acids of the hinge region of human IgG, the hinge region of human IgG comprising a native hinge region or a variant thereof. In some embodiments, the first hinge or / and the second hinge comprises amino acids of the hinge region of human IgG1. In some embodiments, the first hinge or / and the second hinge comprises amino acids of the hinge region of human IgG4. In some embodiments, the first hinge comprises GEPKSSDKTHTCPPCP (SEQ ID NO: 3) and the second hinge comprises EPKSCDKTHTCPPCP (SEQ ID NO: 4). In another embodiment, the first hinge comprises EPKSCDKTHTCPPCP and the second hinge comprises GEPKSSDKTHTCPPCP. In some embodiments, both the first hinge and the second hinge comprise GEPKSSDKTHTCPPCP or EPKSCDKTHTCPPCP.

[0133] Fc area The Fc region of a multispecific antibody is composed of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc region of an immunoglobulin G (IgG) molecule is a dimer, with each Fc polypeptide comprising the CH2 and CH3 of the IgG heavy chain constant region. The two Fc polypeptides of an Fc region can stably associate with each other. In one embodiment, the multispecific antibody of the present invention comprises one Fc region.

[0134] In one embodiment, the Fc region of the multispecific antibody is an IgG Fc region. In one embodiment, the Fc region is an IgG1 Fc region. In another embodiment, the Fc region is an IgG4 Fc region. In a more specific embodiment, the Fc region is an IgG4 Fc region that comprises an amino acid substitution at position S228, in particular the amino acid substitution S228P, which reduces in vivo Fab arm exchange of IgG4 antibodies. In another embodiment, the Fc region is a human Fc region. In a more specific embodiment, the Fc region is a human IgG 1 Fc region.

[0135] In some embodiments, the Fc region comprises modifications, such as amino acid substitutions, including, for example, modifications that promote heterodimerization, modifications that alter effector function, modifications that alter binding ability to Protein A, and the like.

[0136] In some embodiments, the Fc comprises a modification that promotes heterodimerization.

[0137] Since the multispecific antibodies of the present invention comprise different antigen-binding moieties fused to one or the other of the two Fc polypeptides in the Fc region, the two Fc polypeptides are usually comprised in two different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of the multispecific antibodies in recombinant production, it is preferred to introduce modifications in the Fc region of the multispecific antibodies that promote the association of the desired polypeptides. Thus, in a specific embodiment, the Fc region comprises amino acid substitutions that promote the association of the two Fc polypeptides of the Fc region.

[0138] The site of the most extensive protein-protein interaction between two Fc polypeptides of a human IgG Fc region is in the CH3 domain of the Fc region, therefore, in one embodiment, the modification is in the CH3 domain of the Fc region.

[0139] In a specific embodiment, the modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two Fc polypeptides of the Fc region and a "hole" modification in the other of the two Fc polypeptides of the Fc region. Typically, this method promotes heterodimer formation and discourages homodimer formation by introducing a protrusion ("knob") into the contact surface of one Fc polypeptide and a corresponding cavity ("hole") into the contact surface of the other Fc polypeptide and positioning the protrusion in the cavity. The protrusion is constructed by replacing a small amino acid side chain from the contact surface of one Fc polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protrusion is created in the contact surface of the other Fc polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0140] Thus, in a specific embodiment, an amino acid residue in the CH3 domain of one of the Fc polypeptides of the multispecific antibody is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the CH3 domain of that Fc polypeptide that can be positioned in a cavity in the CH3 domain of the other Fc polypeptide. An amino acid residue in the CH3 domain of the other Fc polypeptide is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of that Fc polypeptide.

[0141] In some embodiments, one Fc polypeptide of the Fc region comprises T366Y / W or / and S354C, and the other Fc polypeptide comprises Y407T / V, Y349C, T366S, or / and L368A. As a more specific example, one of the Fc polypeptides in the Fc region comprises amino acid substitutions T366Y / W and S354C, and the other Fc polypeptide comprises amino acid substitutions Y407T / V, Y349C, T366S, and L368A. In a more specific example, the Fc can be a human IgG1 Fc.

[0142] In some embodiments, the Fc region comprises a modification that alters effector function. In some embodiments, the Fc region of the multispecific antibody has been modified to reduce the binding affinity and / or effector function of the Fc region to an Fc receptor compared to the Fc prior to modification. Reducing the binding affinity and / or effector function of the Fc region to an Fc receptor is advantageous in improving cytokine release and side effects.

[0143] In some embodiments, the modification that reduces the binding affinity and / or effector function of the Fc region to an Fc receptor is an amino acid substitution. In some embodiments, the Fc region comprises an amino acid substitution at one or more positions selected from E233, L234, L235, N297, P331 and P329. In some embodiments, the Fc region comprises an amino acid substitution at one or more positions selected from L234, L235 and P329. In some embodiments, the Fc region comprises the amino acid substitutions L234A and L235A. In one such embodiment, the Fc is an IgG1 Fc, in particular a human IgG1 Fc. In some embodiments, the Fc region comprises an amino acid substitution at position P329. In more specific embodiments, the amino acid substitution is P329A, P329R or P329G. In some embodiments, the Fc region comprises an amino acid substitution at position P329 and another amino acid substitution at a position selected from E233, L234, L235, N297, and P331. In more specific embodiments, the other amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In some embodiments, the Fc region comprises amino acid substitutions at positions P329, L234, and L235. In a more specific embodiment, the Fc region comprises amino acid substitutions L234A, L235A, and P329G ("P329G LALA"). In another more specific embodiment, the Fc region comprises amino acid substitutions L234A, L235A, and P329A ("P329A LALA"). In one such embodiment, the Fc is an IgG1 Fc, in particular a human IgG1 Fc.

[0144] In some embodiments, the Fc region is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329A / G / R.

[0145] The amino acid substitutions that reduce the binding affinity and / or effector function of the Fc region to the Fc receptors described above occur in the two polypeptide chains of the Fc region.

[0146] In some embodiments, the Fc region comprises a modification that reduces or eliminates binding between the CH3 region of one Fc polypeptide in the Fc region and Protein A (Protein A from Staphylococcus aureus). In some embodiments, the modification is an amino acid substitution. In some embodiments, the Fc region comprises the amino acid substitutions H435R and / or Y436F, which occur only in one Fc polypeptide and not in the other Fc polypeptide. In one embodiment, the Fc region comprises the amino acid substitutions H435R and / or Y436F, which occur only in one Fc polypeptide. In one such embodiment, the Fc is an IgG1 Fc, particularly a human IgG1 Fc.

[0147] In the multispecific antibody of the present invention, the Fc region may comprise one, two or three of a modification that promotes heterodimerization, a modification that alters effector function, and a modification that reduces or eliminates binding between the CH3 region of one Fc polypeptide in the Fc and Protein A. In some embodiments, the Fc comprises a modification that promotes association of two Fc polypeptides, a modification that reduces the binding affinity and / or effector function of the Fc region for the Fc receptor, and a modification that reduces or eliminates binding between the CH3 region of one Fc polypeptide in the Fc and Protein A. Specific forms of each of the above types of modifications can be combined. For example, in one embodiment, the Fc region comprises, as amino acid substitutions, i. Y407T / V, Y349C, T366S, L368A, T366Y / W and S354C, where the amino acid substitutions T366Y / W and S354C are in the same Fc polypeptide and are in a separate Fc polypeptide from the other amino acid substitutions in (i.), ii. L234A, L235A and P329A; iii. Contains H435R and Y436F, which occur in only one Fc polypeptide.

[0148] In a more specific embodiment, based on EU numbering, one of the Fc polypeptides in the Fc region 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 such an embodiment, the Fc is an IgG1 Fc, particularly a human IgG1 Fc.

[0149] In the present description, amino acid substitutions are indicated in the form of original amino acid-position-substituted amino acid, with the amino acid residue being indicated by a three-letter code (Xaa) or a one-letter code (X). Thus, for example, "H435R" means that the amino acid H at position 435 is replaced by the amino acid R. Multiple replacement amino acids may be included, for example, T366Y / W means that the amino acid T at position 366 is replaced by the amino acid Y or W.

[0150] In some embodiments, the multispecific antibodies according to the invention are trivalent, i.e., the first antigen-binding moiety, the second antigen-binding moiety and the third antigen-binding moiety all provide monovalent binding to the corresponding antigen.

[0151] The present invention provides examples of trivalent multispecific antibodies.

[0152] In one example, the multispecific antibody is composed of two polypeptide chains represented by SEQ ID NO: 65 and SEQ ID NO: 67, respectively. In some embodiments, the nucleotide sequences encoding the two polypeptide chains are represented by SEQ ID NO: 66 and SEQ ID NO: 68, respectively.

[0153] In one example, the multispecific antibody is composed of two polypeptide chains represented, respectively, by SEQ ID NO: 69 and SEQ ID NO: 71. In some embodiments, the nucleotide sequences encoding the two polypeptide chains are represented, respectively, by SEQ ID NO: 70 and SEQ ID NO: 72.

[0154] As an example, a multispecific antibody is composed of three polypeptide chains represented, respectively, by SEQ ID NO: 59, SEQ ID NO: 61 and SEQ ID NO: 63. In some embodiments, the nucleotide sequences encoding the two polypeptide chains are represented, respectively, by SEQ ID NO: 60, SEQ ID NO: 62 and SEQ ID NO: 64.

[0155] As an example, the multispecific antibody is composed of three polypeptide chains represented, respectively, by SEQ ID NO: 73, SEQ ID NO: 61 and SEQ ID NO: 75. In some embodiments, the nucleotide sequences encoding the two polypeptide chains are represented, respectively, by SEQ ID NO: 74, SEQ ID NO: 62 and SEQ ID NO: 76.

[0156] composition The invention provides pharmaceutical compositions comprising a multispecific antibody and one or more pharma- ceutically acceptable carriers, such as excipients, diluents, encapsulating materials, fillers, buffers, or other additives.

[0157] Isolated Nucleic Acids The present invention provides isolated nucleic acids, including polynucleotides, encoding the multispecific antibodies according to the invention. The nucleic acid sequences of some of the multispecific antibodies are illustrated in the Sequence Listing.

[0158] vector The present invention provides a vector comprising the nucleic acid. In some embodiments, the vector is a cloning vector. In other embodiments, the vector is an expression vector. As a specific example, the expression vector is pcDNA3.1(+). The expression vector can be any expression vector capable of expressing the multispecific antibody described herein.

[0159] host cell In some embodiments, the present invention provides a host cell comprising the vector of the present invention. The host cell is a suitable host cell for cloning or expressing a multispecific antibody. 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 a yeast cell, a mammalian cell, or other cells suitable for producing a multispecific antibody. Mammalian cells include, for example, Chinese Hamster Ovary (CHO) cells, CHO-S cells.

[0160] Methods for producing multispecific antibodies In some embodiments, the present invention provides a method for producing a multispecific antibody, comprising culturing the host cell to express the multispecific antibody, and isolating and purifying the multispecific antibody from the system. To produce the multispecific antibody, nucleic acids encoding the multispecific antibody are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. The nucleic acids can be obtained by various methods well known in the art, such as gene splicing, chemical synthesis, etc.

[0161] use The present invention provides for the use of multispecific antibodies. In some embodiments, the multispecific antibody used may be BC24, PC1, PC2 and / or PC3.

[0162] The present invention provides the use of said multispecific antibody or said pharmaceutical composition for the manufacture of a medicament for treating a disease associated with BCMA expression.

[0163] The present invention provides a method for treating a disease associated with BCMA expression in a subject, comprising administering to the subject a therapeutically effective amount of said multispecific antibody or said pharmaceutical composition.

[0164] In some embodiments, the disease is a tumor or an autoimmune disease.

[0165] In some embodiments, the disease is lymphoma, multiple myeloma, leukemia, systemic lupus erythematosus, or rheumatoid arthritis.

[0166] The present invention also provides example embodiments.

[0167] [Embodiment 1] (i) a first antigen-binding portion that binds to a first antigen; and (ii) a second antigen-binding portion that binds to a second antigen; and (iii) a third antigen-binding moiety that binds to the first antigen, the first antigen is BCMA and the second antigen is a T cell activation antigen, and the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains, each independently comprising: (a) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9; (b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or (d) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18 A multispecific antibody comprising any one of the following:

[0168] [Embodiment 2] The first antigen-binding portion and the third antigen-binding portion each independently comprise: (b) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, or (c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15 2. The multispecific antibody of embodiment 1, comprising any one of:

[0169] [Embodiment 3] The first antigen-binding portion comprises, as complementarity determining regions, CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and The multispecific antibody of embodiment 2, wherein the third antigen-binding portion comprises, as complementarity determining regions, CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15.

[0170] [Embodiment 4] The third antigen-binding portion comprises, as complementarity determining regions, CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and The multispecific antibody of embodiment 2, wherein the first antigen-binding portion comprises, as complementarity determining regions, CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15.

[0171] [Embodiment 5] The first antigen-binding portion and the third antigen-binding portion each independently comprise CDR1, CDR2 and CDR3 of a single variable domain comprising an amino acid sequence represented by SEQ ID NO: 19, 21, 23, 25, 37, 38, 39 or 40, preferably, the first antigen-binding portion and the third antigen-binding portion each independently comprise CDR1, CDR2 and CDR3 of a single variable domain comprising an amino acid sequence represented by SEQ ID NO: 37 or 39; 2. The multispecific antibody of embodiment 1.

[0172] [Embodiment 6] The multispecific antibody according to any one of embodiments 1 to 5, wherein the first antigen-binding portion and the third antigen-binding portion each independently comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence represented by SEQ ID NO: 19, 41, 42 or 25.

[0173] [Embodiment 7] 7. The multispecific antibody according to any one of embodiments 1 to 6, wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:41.

[0174] [Embodiment 8] 8. The multispecific antibody of embodiment 7, wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 21, 37 or 38, preferably wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 37.

[0175] [Embodiment 9] The multispecific antibody according to any one of embodiments 1 to 8, wherein the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:42.

[0176] [Embodiment 10] 10. The multispecific antibody of embodiment 9, wherein the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 23, 39 or 40, preferably wherein the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 39.

[0177] [Embodiment 11] 7. The multispecific antibody according to any one of embodiments 1 to 6, wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:42.

[0178] [Embodiment 12] 12. The multispecific antibody of embodiment 11, wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 23, 39 or 40, preferably wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 39.

[0179] [Embodiment 13] The multispecific antibody according to any one of embodiments 1 to 6, and 11 to 12, wherein the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO:41.

[0180] [Embodiment 14] 14. The multispecific antibody of embodiment 13, wherein the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 21, 37 or 38, preferably wherein the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 37.

[0181] [Embodiment 15] The first antigen-binding portion and the third antigen-binding portion are (1) a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO:21 and a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO:23, 39, or 40; (2) a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 37 and a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (3) a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 38 and a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (4) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 21 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (5) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 37 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (6) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 38 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (7) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 41 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 42; (8) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 42 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 41; (9) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 37 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 39; or (10) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 39 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 37; The multispecific antibody according to any one of embodiments 1 to 5, which is any one selected from the following:

[0182] [Embodiment 16] 16. The multispecific antibody according to any one of embodiments 1 to 15, wherein said single variable domains are camelid or humanized.

[0183] [Embodiment 17] 17. The multispecific antibody according to any one of embodiments 1 to 16, wherein the first antigen-binding portion and the third antigen-binding portion are the same.

[0184] [Embodiment 18] 17. The multispecific antibody according to any one of embodiments 1 to 16, wherein the first antigen-binding portion and the third antigen-binding portion are different.

[0185] [Embodiment 19] The multispecific antibody of any one of embodiments 1 to 18, wherein the second antigen-binding portion is a Fab, ScFv or ScFab.

[0186] [Embodiment 20] The multispecific antibody according to any one of embodiments 1 to 19, wherein the T cell activation antigen is CD3.

[0187] [Embodiment 21] The second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises, as complementarity determining regions, HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 43, HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, and the light chain variable region comprises LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 48. The multispecific antibody according to any one of embodiments 1 to 20.

[0188] [Embodiment 22] The multispecific antibody according to any one of embodiments 1 to 20, wherein the second antigen-binding portion 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 represented by SEQ ID NO: 49, and the light chain variable region comprising LCDR1, HCDR2 and LCDR3 of the variable region represented by SEQ ID NO: 50.

[0189] [Embodiment 23] 23. The multispecific antibody according to any one of embodiments 1 to 22, wherein the second antigen-binding portion comprises a heavy chain variable region having an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 49 and a light chain variable region having an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 50, and preferably the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence represented by SEQ ID NO: 49 and the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 50.

[0190] [Embodiment 24] 24. The multispecific antibody of any one of embodiments 1 to 23, wherein the second antigen-binding portion is murine, chimeric or humanized.

[0191] [Embodiment 25] 25. The multispecific antibody of any one of embodiments 1 to 24, wherein the third antigen-binding portion and the first antigen-binding portion are fused to each other, optionally fused to each other via a peptide linker.

[0192] [Embodiment 26] 26. The multispecific antibody of embodiment 25, wherein said third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion.

[0193] [Embodiment 27] 25. The multispecific antibody of any one of embodiments 1 to 24, wherein the third antigen-binding portion and the second antigen-binding portion are fused to each other, optionally fused to each other via a peptide linker.

[0194] [Embodiment 28] 28. The multispecific antibody of embodiment 27, wherein said third antigen-binding portion is fused at its C-terminus to the N-terminus of the second antigen-binding portion.

[0195] [Embodiment 29] (iv) The multispecific antibody according to any one of embodiments 1 to 28, further comprising an Fc region composed of two Fc polypeptides.

[0196] [Embodiment 30] 30. The multispecific antibody of embodiment 29, wherein the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region and the second antigen-binding portion is an ScFv and the second antigen-binding portion is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region.

[0197] [Embodiment 31] 31. The multispecific antibody of embodiment 30, wherein said second antigen-binding portion has the structure VL-VH or VH-VL from N-terminus to C-terminus.

[0198] [Embodiment 32] 30. The multispecific antibody of embodiment 29, wherein the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region and the second antigen-binding portion is a Fab and the second antigen-binding portion is fused at the C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region.

[0199] [Embodiment 33] 33. The multispecific antibody of embodiment 32, wherein the third antigen-binding portion is fused to the N-terminus of the Fab light chain or Fab heavy chain of the second antigen-binding portion.

[0200] [Embodiment 34] 30. The multispecific antibody of embodiment 29, wherein the second antigen-binding portion is an ScFv, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion.

[0201] [Embodiment 35] 30. The multispecific antibody of embodiment 29, wherein the second antigen-binding portion is an ScFv, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the second antigen-binding portion.

[0202] [Embodiment 36] 30. The multispecific antibody of embodiment 29, wherein the second antigen-binding portion is a Fab, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused at the C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion.

[0203] [Embodiment 37] 30. The multispecific antibody of embodiment 29, wherein the second antigen-binding portion is a Fab, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide of the Fc region, the second antigen-binding portion is fused at its C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide of the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of a Fab light chain of the second antigen-binding portion or the N-terminus of a Fab heavy chain of the second antigen-binding portion.

[0204] [Embodiment 38] The multispecific antibody according to any one of embodiments 29 to 37, wherein the Fc region is an IgG Fc region.

[0205] [Embodiment 39] 39. The multispecific antibody according to embodiment 38, wherein said IgG Fc region is a human IgG Fc region, preferably a human IgG1 or human IgG4 Fc region.

[0206] [Embodiment 40] The multispecific antibody of any one of embodiments 29 to 39, wherein said Fc region comprises an amino acid substitution that promotes association of the two Fc polypeptides of said Fc region.

[0207] [Embodiment 41] 41. The multispecific antibody according to any one of embodiments 29 to 40, wherein, based on EU numbering, one Fc polypeptide in said Fc region comprises the amino acid substitutions T366Y / W and S354C and the other Fc polypeptide comprises the amino acid substitutions Y407T / V, Y349C, T366S and L368A.

[0208] [Embodiment 42] The multispecific antibody according to any one of embodiments 29 to 41, wherein the Fc region comprises an amino acid substitution that reduces the binding affinity of the Fc region for an Fc receptor and / or an effector function.

[0209] [Embodiment 43] 43. The multispecific antibody of embodiment 42, wherein, based on EU numbering, the amino acid substitutions that reduce the binding affinity of the Fc region to an Fc receptor and / or effector function are at one or more positions selected from L234, L235 and P329, and preferably both Fc polypeptides of said Fc region comprise the amino acid substitutions L234A, L235A and P329G; or both comprise the amino acid substitutions L234A, L235A and P329A.

[0210] [Embodiment 44] The multispecific antibody according to any one of embodiments 29 to 41, wherein the Fc region comprises an amino acid substitution that reduces or eliminates binding between the CH3 region of one Fc polypeptide in the Fc region and Protein A.

[0211] [Embodiment 45] 45. The multispecific antibody of embodiment 44, wherein, based on EU numbering, said Fc region comprises the amino acid substitutions H435R or / and Y436F, which occur in only one Fc polypeptide.

[0212] [Embodiment 46] 40. The multispecific antibody according to any one of embodiments 29 to 39, wherein in said Fc region, one Fc polypeptide comprises the amino acid substitutions L234A, L235A, P329A, Y349C, T366S, L368A and Y407V and the other Fc polypeptide comprises the amino acid substitutions L234A, L235A, P329A, S354C, T366W, H435R and Y436F, based on EU numbering.

[0213] [Embodiment 47] The multispecific antibody according to any one of embodiments 1 to 46, which is trivalent.

[0214] [Embodiment 48] (1) a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 65, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 67; (2) a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO:69, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO:71; (3) A polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 59, a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 61, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 63; or (4) A polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 73, a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 61, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 75. The multispecific antibody according to any one of embodiments 1 to 46, wherein

[0215] [Embodiment 49] (1) A polypeptide chain comprising two polypeptide chains, one containing the amino acid sequence represented by SEQ ID NO:65 and the other containing the amino acid sequence represented by SEQ ID NO:67; (2) a polypeptide chain comprising two polypeptide chains, one containing the amino acid sequence represented by SEQ ID NO:69 and the other containing the amino acid sequence represented by SEQ ID NO:71; (3) A polypeptide chain comprising an amino acid sequence represented by SEQ ID NO: 59, an amino acid sequence represented by SEQ ID NO: 61, and an amino acid sequence represented by SEQ ID NO: 63; or (4) A polypeptide chain comprising three polypeptide chains, one comprising the amino acid sequence represented by SEQ ID NO: 73, one comprising the amino acid sequence represented by SEQ ID NO: 61, and one comprising the amino acid sequence represented by SEQ ID NO: 75. 2. The multispecific antibody of embodiment 1, wherein

[0216] [Embodiment 50] An isolated nucleic acid comprising a polynucleotide encoding the multispecific antibody according to any one of embodiments 1 to 49.

[0217] [Embodiment 51] A vector comprising the nucleic acid described in embodiment 50.

[0218] [Embodiment 52] A host cell comprising a nucleic acid according to embodiment 50 or a vector according to embodiment 51.

[0219] [Embodiment 53] A method for producing a multispecific antibody according to any one of Embodiments 1 to 49, comprising culturing the host cell according to Embodiment 52 to express the multispecific antibody, and isolating and purifying the multispecific antibody from the system.

[0220] [Embodiment 54] A pharmaceutical composition comprising the multispecific antibody according to any one of embodiments 1 to 49 and a pharma- ceutically acceptable carrier.

[0221] [Embodiment 55] A method for treating a disease associated with BCMA expression in a subject, comprising administering to said subject a therapeutically effective amount of a multispecific antibody according to any one of embodiments 1 to 49 or the pharmaceutical composition according to embodiment 54.

[0222] [Embodiment 56] 56. The method of embodiment 55, wherein the disease is lymphoma, multiple myeloma, leukemia, systemic lupus erythematosus, or rheumatoid arthritis.

[0223] [Embodiment 57] (1) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9; (2) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (3) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, or (4) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 18; A BCMA binding antibody comprising a single variable domain comprising:

[0224] [Embodiment 58] 58. The BCMA-binding antibody of embodiment 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 to a sequence set forth in SEQ ID NO: 19, 21, 23, 25, 37, 38, 39, 40, 41 or 42.

[0225] [Embodiment 59] 59. The BCMA-binding antibody of embodiment 58, wherein the single variable domain comprises an amino acid sequence represented by SEQ ID NO: 37, 38, 39 or 40.

[0226] [Embodiment 60] 60. The BCMA-binding antibody of any one of embodiments 57 to 59, wherein the single variable domain is camelid or humanized.

[0227] [Embodiment 61] 61. The BCMA-binding antibody of any one of embodiments 57 to 60, comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 27, 29, 31, 33, 51, 53, 55 or 57. EXAMPLES

[0228] Example 1: Construction of an anti-BCMA single domain antibody phage display library Bactrian camels were immunized by subcutaneous multiple injections of recombinant human BCMA-Fc fusion protein (ACRO, Cat. No. BC7-H5254) mixed and emulsified in Freund's complete adjuvant at a volume ratio of 1:1, followed by booster immunizations every 2 weeks with recombinant human BCMA-Fc fusion protein mixed and emulsified in Freund's incomplete adjuvant at a volume ratio of 1:1. After the fourth or fifth immunization, serum was collected and anti-human BCMA antibody titers were measured. After multiple immunizations, peripheral blood was collected from the Bactrian camels and peripheral blood mononuclear cells (PBMCs) were isolated. RNA from PBMCs was extracted and reverse transcribed into cDNA, and the V of camel antibodies was analyzed. H The H coding fragment was amplified.

[0229] Amplified V H The H-encoding fragment was treated with PstI / NotI endonuclease and then inserted into the phagemid vector pMECS (NTCC Plasmid Vector Cultured Cell Gene Depository Center, Catalog No. pMECS) to construct a recombinant vector, which was then electroporated into E. coli TG1 (Lucigen, Catalog No. 60502-1) to construct the library expression strain. H The H expression strain was cultured to logarithmic growth phase, and the library was amplified by adding helper phage M13KO7 (New England Biolabs, catalog number N0315S), and shaken at 28°C, 200 rpm overnight. The bacterial solution was centrifuged to remove the supernatant, and 1 / 4 volume of PEG6000 / NaCl solution (20% PEG6000 (w / v), 2.5M NaCl) was added to the supernatant, and the phage was precipitated by incubation on ice for 1 to 2 hours. The phage precipitate was collected by centrifugation, resuspended in PBS, and then added with 20% glycerol. The resulting mixture was stored at -80°C as a single domain antibody phage display library.

[0230] Example 2: Screening of anti-human BCMA single domain antibodies A single domain antibody phage display library was panned by solid-phase panning. Single clones obtained by panning were cultured and induced with isopropyl-β-D-thiogalactopyranoside (IPTG), and the supernatant was prepared.

[0231] The selected clones were tested positive by indirect ELISA against human BCMA-His (ACRO, Cat. No. BCA-H522y). Positive clones that bound only to human BCMA-His and had relatively high signal values ​​were selected, stored, and sequenced. Positive clones 1A1, 1A10, 1A11, and 1B10 were selected. Sequence analysis revealed that 1A1 was V H The H nucleotide sequence was SEQ ID NO:20 and the amino acid sequence was SEQ ID NO:19. H The H nucleotide sequence was SEQ ID NO:22 and the amino acid sequence was SEQ ID NO:21. H The H nucleotide sequence was SEQ ID NO:24 and the amino acid sequence was SEQ ID NO:23. H The H nucleotide sequence was SEQ ID NO:26 and the amino acid sequence was SEQ ID NO:25.

[0232] Example 3: Anti-human BCMA V H Preparation of H-Fc chimeric antibody 3.1 V H Preparation of H-Fc chimeric antibody V of selected positive clones H The H sequence is linked to a human Fc region and the V H A H-Fc chimeric antibody was constructed. H H sequence or anti-human BCMA V H The H control antibody (BM) sequence (amino acid sequence is the same as SEQ ID NO: 125 of CN109153731A) was inserted into the pCDNA3.1 eukaryotic expression vector containing the human IgG1 constant region (amino acid sequence is SEQ ID NO: 5) and expressed using the transient expression system Expifectamine (商標) These VH H-Fc chimeric antibody (BM-Fc is a control) was expressed. After purification by a protein A affinity column, the binding status of the antibody with human TACI and BAFF-R proteins was analyzed by surface plasmon resonance (see 3.2 for the method). As a result of the analysis, 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc were all able to specifically bind to BCMA-His protein, but were unable to bind to human TACI and BAFF-R proteins. As a result of sequence analysis, the full-length amino acid sequence of 1A1-Fc was as shown in SEQ ID NO:27, and the nucleotide sequence was as shown in SEQ ID NO:28. The full-length amino acid sequence of 1A10-Fc was as shown in SEQ ID NO:29, and the nucleotide sequence was as shown in SEQ ID NO:30. The full-length amino acid sequence of 1A11-Fc was as shown in SEQ ID NO:31, and the nucleotide sequence was as shown in SEQ ID NO:32. 1B10-Fc had a full-length amino acid sequence as set forth in SEQ ID NO:33 and a nucleotide sequence as set forth in SEQ ID NO:34.

[0233] 3.2 Analysis of antibody binding to human TACI and BAFF-R proteins by surface plasmon resonance Anti-human BCMA V was detected using a biomolecular interaction analysis system (GE, Biacore T200). H The specificity of the H-Fc chimeric antibody was tested. Anti-hIgG(Fc) Antibody (GE, Cat. No. BR-1008-39) was amine-coupled to the sensor chip CM5, and anti-human BCMA V was HThe H-Fc chimeric antibody was diluted to 2 μg / ml in 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) and captured through the experimental channel for 90 s at a flow rate of 30 μl / min. Human TACI or BAFF-R protein was diluted to 100 nM in running buffer and allowed to bind at a flow rate of 50 μl / min, and binding signal curves were observed. No binding curves were observed for 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc. None of them bound to TACI or BAFF-R protein.

[0234] Example 4: Anti-human BCMA V H Affinity of H-Fc chimeric antibody to human-cynomolgus BCMA 4.1 Measurement of affinity of antibodies to human and cynomolgus BCMA by surface plasmon resonance Anti-human BCMA V was detected using a biomolecular interaction analysis system (GE, Biacore T200). H The affinity of the H-Fc chimeric antibodies was tested. As a result, 1A1-Fc, 1A10-Fc, 1A11-Fc, and 1B10-Fc all had high affinity for human BCMA protein, and among them, 1A10-Fc and 1A11-Fc showed cross-reactivity to cynomolgus monkey BCMA protein, but 1A1-Fc, 1B10-Fc, and BM-Fc showed no binding to cynomolgus monkey BCMA protein.

[0235] 4.2 Measurement of antibody-cell binding by flow cytometry Flow cytometry revealed that anti-human BCMA V HThe binding of H-Fc chimeric antibodies to target cells with different BCMA expression levels was confirmed, where CHO-hBCMA cells (Akangde Biomedical Technology (Suzhou) Co., Ltd., Catalog No. AKD001A) are a stable cell line with high BCMA expression, U266 cells (Center for Basic Medical Cells, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Catalog No. 3111C0001CCC000684) are natural human myeloma cell lines with moderate BCMA expression, PRMI8226 cells (Center for Basic Medical Cells, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Catalog No. 3111C0001CCC000083) are natural human myeloma cell lines with low BCMA expression, and HUVEC cells (ScienCell Research Laboratories, Catalog No. AKD001A 8000) are human umbilical vein endothelial cell lines that do not express BCMA. Anti-human BCMA V diluted in a gradient (initial concentration 126 nM, 5-fold gradient dilution, 6 concentrations) H H-Fc chimeric antibody: 2 × 10 5 The target cells were incubated and incubated on ice for 1 h, then the cells were washed and PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, Cat. No. 109-116-170) was added and incubated on ice for 0.5 h. The cells were washed and then analyzed by flow cytometer (Thermo Fisher Scientific Inc., Attune NXT).

[0236] As shown in Figure 1A-1C and Table 1, 1A1-Fc, 1A10-Fc, 1A11-Fc and 1B10-Fc could effectively target and bind to CHO-hBCMA cells with high BCMA expression and U266 cells with moderate BCMA expression, and also showed clear binding to RPMI8226 cells with low BCMA expression. 1A10-Fc and 1A11-Fc showed better binding to U266 cells than BM-Fc. As shown in Figure 1D, 1A10-Fc and 1A11-Fc did not show binding to HUVEC cells that do not express BCMA, but 1A1-Fc and 1B10-Fc showed binding to HUVEC cells that do not express BCMA.

[0237] [Table 6]

[0238] 4.3 Measurement of antibody binding to cynomolgus BCMA transient HEK293T cells by flow cytometry The full-length cynomolgus BCMA gene (SEQ ID NO: 6) was synthesized in vitro and inserted into the pCDNA3.1 eukaryotic expression vector using Lipofectamine according to the manufacturer's protocol. (商標) The expression vector was transfected into HEK293T cells using Thermo Fisher Scientific Inc. Cat. No. L3000015. Briefly, HEK293T cells were transfected at 5 × 10 5 Cells were seeded at 1000 x 1000 / well in a 6-well plate. After approximately 24 hours, the medium containing fetal bovine serum was aspirated, the cells were washed with PBS, and the transfection reagent was added to each well at a ratio of DNA:Lipo3000=5μg:7.5μl. After approximately 6 hours, the medium was replaced with one containing 10% fetal bovine serum (v / v). Approximately 24 hours after transfection, the cells were analyzed by flow cytometry.

[0239] 1A10-Fc, 1A11-Fc and BM-Fc antibodies were diluted in a gradient (initial concentration 126 nM, 5-fold gradient dilution, 5 concentrations, sample dilution was used as a negative control) and incubated with 2 × 10 cells transiently transfected with cynomolgus monkey BCMA and untransfected HEK293T cells. 5After incubation for 1 hour on ice, the cells were washed and incubated with PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, Catalog No. 109-116-170) for 0.5 hours on ice, the cells were washed, and then analyzed with a flow cytometer (Thermo Fisher Scientific Inc., Attune NXT). A in Figure 2 shows HEK293T cells transiently transfected with cynomolgus BCMA (HEK293T-CynoBCMA), and B in Figure 2 shows HEK293T cells not transfected with cynomolgus BCMA. Table 2 shows the EC50 value and maximum binding MFI value (Bmax) of the binding between the Fc chimeric antibody and HEK293T cells transiently transfected with cynomolgus BCMA. The results showed that 1A10-Fc and 1A11-Fc bound to cynomolgus BCMA on cells, whereas the BM-Fc antibody did not bind to cynomolgus BCMA.

[0240] [Table 7]

[0241] Example 5: Anti-human BCMA V H Blockade of APRIL-BCMA binding by H-Fc chimeric antibodies Anti-human BCMA V in serial dilutions (initial concentration 252 nM, 5-fold serial dilutions, 7 concentrations) HEach of the H-Fc chimeric antibodies 1A10-Fc, 1A11-Fc and BM-Fc was mixed with 100 ng / ml of biotin-labeled recombinant BCMA-His protein (ACRO, catalog number BCA-H522y) at a volume ratio of 1:1, incubated at room temperature for 1 hour, and then added to an ELISA plate immobilized with recombinant APRIL protein (ACRO, catalog number APL-H5244). In addition, a control group was prepared by adding only 50 ng / ml of biotin-labeled recombinant BCMA-His protein. After incubation at 37°C for 1 hour, unbound biotin-labeled BCMA-His protein was washed away, HRP-labeled streptavidin (eBioscience, catalog number 18-4100-51) was added, and the plate was washed five times before color development. The absorbance signal values ​​were measured at a wavelength of 450 nm and a reference wavelength of 650 nm. The blocking rate of each antibody sample at each concentration relative to the signal value of BCMA and APRIL binding was calculated using the following formula, and curve fitting was performed.

[0242] Blocking rate = (signal value of control group - signal value of sample) / signal value of control group x 100% As a result, as shown in Figure 3 and Table 3, 1A1-Fc, 1B10-Fc, 1A10-Fc and 1A11-Fc were all able to effectively block the binding of APRIL to BCMA.

[0243] As shown in Figure 4A, the amino acid sequence of human BCMA extracellular domain (SEQ ID NO: 35) and cynomolgus BCMA extracellular domain (SEQ ID NO: 36) differs at Gly6, Ala20, Ile22, Asn31, Val45 and Thr52. Since clones 1A10 and 1A11 showed cross-reactivity with cynomolgus BCMA protein, it is speculated that the possible epitopes of these clones are located at Gln7-His19, and / or Pro23-Ser30, and / or Asn31-Ser44, and / or Thr46-Gly51. Figure 4B shows the structure of the extracellular domain of human BCMA in the PDB database (PDB number: 2kn1). As shown in Figures 4A and 4B, the extracellular domain of human BCMA contains three pairs of disulfide bonds: Cys8-Cys21, Cys24-Cys37, and Cys28-Cys41. Since the main binding between human BCMA and APRIL is in a β-hairpin structure (Bossen, C. et al. Semin. Immunol. 2006, 18(5):263-275), the main binding sites of clones 1A10-Fc and 1A11-Fc are considered to be between Gln7 and His19, and / or between Pro23 and Ser30, and / or between Asn31 and Ser44.

[0244] [Table 8]

[0245] Example 6: Anti-human BCMA V H Analysis of epitope differences among clones of H 6.1 Anti-human BCMA V by ELISA (checkerboard method) H Analysis of competitive relationships between clones of H Anti-human BCMA V H The H-Fc chimeric antibody was diluted to 2 μg / ml and coated onto a high-binding ELISA plate, which was then washed and blocked. HThe H-Fc chimeric antibody and biotin-labeled BCMA-His protein (ACRO, catalog number BCA-H522y) were incubated at room temperature for 0.5 hours to obtain an antigen-antibody mixture. The incubated antibody-antigen mixture or only biotin-labeled BCMA-His protein (control group) was added to a well plate at 100 μl / well according to the checkerboard arrangement method and incubated at 37°C for 1 hour. Unbound biotin-labeled BCMA-His protein was washed away, HRP-labeled streptavidin (eBioscience, catalog number 18-4100-51) was added, and the plate was washed five times before color development. The absorbance signal values ​​at a wavelength of 450 nm and a reference wavelength of 650 nm were measured. The blocking rate of one antibody against the signal of the binding of another antibody to BCMA-Bio was calculated by the following formula.

[0246] Blocking rate = (signal value of control group - signal value of sample) / signal value of control group x 100% As a result, as shown in Table 4, the diagonal line "\" (gray) of the checkerboard is the positive control group for antibody self-competition, and the blocking rates were all 99% or more. 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%, suggesting that there is no clear competitive relationship between 1A10-Fc and 1A11-Fc, and that 1A10-Fc and 1A11-Fc can simultaneously bind to different epitopes on the BCMA protein. Similarly, there was no clear competitive relationship between 1A10-Fc and 1A1-Fc, or between 1A10-Fc and 1B10-Fc, and it is considered that 1A10-Fc and 1A1-Fc can simultaneously bind to different epitopes on the BCMA protein, and 1A10-Fc and 1B10 can simultaneously bind to different epitopes on the BCMA protein. The mutual blocking rates among 1A1-Fc, 1A11-Fc, and 1B10-Fc all exceeded 99%.

[0247] [Table 9]

[0248] 6.2 Analysis of epitope differences using surface plasmon resonance Epitope competition was analyzed using a biomolecular interaction analysis system (GE, Biacore T200). Anti-His Antibody (GE, Catalog No. 28995056) was amine-coupled to the sensor chip CM5, and BCMA-His protein (ACRO, Catalog No. BCA-H522y) was diluted to about 1 μg / ml with running buffer and captured through the experimental channel at a flow rate of 30 μl / min. The capture signal was adjusted to 180 RU to 190 RU by adjusting the binding time. Anti-human BCMA V H H-Fc chimeric antibody V H H-Fc 1 was diluted to 10 μg / ml (saturating concentration; increasing the concentration does not change the binding signal value) in running buffer and injected until the signal reached the platform. After the injection was completed, another anti-human BCMA V H H-Fc chimeric antibody V H H-Fc2 was immediately injected. The antibody binding curve was observed and the binding signal values ​​of the two antibodies were recorded respectively. The change in signal value is shown in Table 5. The signal value when the binding of the 1A10-Fc antibody to BCMA reached saturation was 472.5 RU, and when 1A11-Fc was further injected at this point, the saturation signal value became 579.0 RU, which was equivalent to the saturation signal value of 653.1 RU when 1A11-Fc was injected alone. The reverse was also true, and the accumulated signal values ​​of the forward and reverse injections were similar. Thus, it is believed that 1A10-Fc and 1A11-Fc can bind to different epitopes on the BCMA protein simultaneously.

[0249] [Table 10]

[0250] 6.3 Analysis of epitope differences by flow cytometry Because BCMA protein expressed on the cell membrane may differ from free protein in terms of effective epitope exposure, we further confirmed by flow cytometry whether the two clones, 1A10 and 1A11, could simultaneously bind to BCMA protein on the cell membrane. U266 cells were used as target cells. 2 × 10 5 The target cells were incubated with 20 μg / ml or 10 μg / ml of the antibody 1A10-Fc, 1A11-Fc alone, or a mixture of 20 μg / ml of the antibody 1A10-Fc and 20 μg / ml of the antibody 1A11-Fc in a 1:1 volume ratio, incubated on ice for 1 hour, washed the cells, added PE-labeled anti-human IgG Fc antibody (Jackson Immuno Research, Catalog No. 109-116-170), incubated on ice for 0.5 hours, washed the cells, and then analyzed by a flow cytometer (Thermo Fisher Scientific Inc., Attune NXT). The results are shown in Table 6. When the same antibody was added at 10 μg / ml, the average fluorescence increase rate of the 1A10-Fc sample was 902.0 / 823.5-1 = 9.5%, and the average fluorescence increase rate of the 1A11-Fc sample was 702.0 / 697.5-1 = 0.6%, so 10 μg / ml is considered to be close to the saturation concentration for both antibodies. On the other hand, when another antibody was added at 10 μg / ml, that is, when 10 μg / ml of 1A11-Fc was added to 10 μg / ml of 1A10-Fc, the average fluorescence increase rate was 1443.5 / 823.5-1 = 75.3%, and when 10 μg / ml of 1A10-Fc was added to 10 μg / ml of 1A11-Fc, the average fluorescence increase rate was 1443.5 / 697.5-1 = 107.0%. Thus, it is believed that the two antibodies 1A10-Fc and 1A11-Fc can simultaneously bind to different epitopes of the BCMA protein on the cell membrane.

[0251] [Table 11]

[0252] Example 7: Anti-human BCMA V HConstruction, expression, and purification of H-Fc chimeric monoclonal antibody and its humanized monoclonal antibody V H The H-Fc chimeric antibodies 1A10-Fc and 1A11-Fc were humanized, respectively. Among them, two humanized antibodies, 1A10-V1 and 1A10-V2, were obtained by humanizing the chimeric antibody 1A10-Fc. Two humanized antibodies, 1A11-V1 and 1A11-V2, were obtained by humanizing the chimeric antibody 1A11-Fc. The nucleotide and amino acid sequences of the above antibodies are shown in Table 7.

[0253] Anti-human BCMA V H DNA sequences encoding the H-Fc chimeric antibody and its humanized antibody (SEQ ID NOs: 30, 52, 54, 32, 56, and 58; corresponding names are shown in Table 7) were synthesized and cloned into the pcDNA3.1(+) expression vector, respectively. Each antibody was expressed using the ExpiCHO expression kit (Thermo Fisher, catalog number A29133). First, the expression vector containing the constructed antibody DNA sequence was transfected into ExpiCHO cells (CHO-S, Thermo). Next, the cells were cultured in ExpiCHO expression medium on an orbital shaker rotating at 130 rpm in a humid atmosphere incubator containing 8% CO2 at 37°C. The culture supernatant was collected, and protein purification was performed using protein A magnetic beads (Genscript, catalog number L00273). Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).

[0254] [Table 12-1]

[0255] [Table 12-2]

[0256] Example 8: Anti-human BCMA V HBinding of H-Fc chimeric antibody and its humanized antibody to the antigen human BCMA The affinity of the test antibodies for human BCMA protein was measured using Biacore T200 (GE) as follows.

[0257] A fixed amount of the test antibodies (including 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, and 1A11-V2) was captured using a chip CM5 (Cytiva, catalog 29149603) coupled with anti-hIgG (Cytiva, catalog 29234600), and then human BCMA was flowed over the chip surface, and the reaction signal was detected in real time using the software Biacore to obtain binding and dissociation curves. The buffer used in the experiment was Biacore general-purpose 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). The reaction value of Anti-hIgG coupled to the surface of chip CM5 reached about 9000RU, and about 200RU of test antibodies (including 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, 1A11-V2) were captured, and then the signal value of the interaction between each concentration of human BCMA (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) and the test antibodies (including 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, 1A11-V2) was measured. The flow rate of the flow cell was 50 μl / min, the binding time was 240 s, the dissociation time was 1400 s, and the regeneration with 3M MgCl2 (GE) was 60 s, with a stable baseline. The results were calculated using the affinity and kinetics 1:1 binding model of the biacore evaluation software. Anti-human BCMA V H The affinity of the H-Fc chimeric antibody and its humanized antibodies (including 1A10-Fc, 1A11-Fc, 1A10-V1, 1A10-V2, 1A11-V1, and 1A11-V2) for human BCMA is shown in Table 8.

[0258] [Table 13]

[0259] Example 9. Construction, expression and purification of anti-BCMA / anti-CD3 multispecific antibodies An anti-BCMA / anti-CD3 multispecific antibody was generated as a human IgG1 by Fc engineering using knobs-into-holes (Ridgway, et al., 1996) technology. One of the polypeptide sequences in the Fc region was designed to contain H435R, Y436F mutations (Jendeberg et al., 1997) that reduce the affinity of the Fc for Protein A, as this is suitable for removing homodimers formed during construction of the multispecific antibody by Protein A affinity purification.

[0260] In this example, four anti-BCMA / anti-CD3 multispecific antibodies of the present invention were constructed, and named BC24, PC1, PC2, and PC3, respectively. The structure of BC24 is shown in Figure 6A, the structure of PC1 is shown in Figure 6B, the structure of PC2 is shown in Figure 6C, and the structure of PC3 is shown in Figure 6D. In the constructed multispecific antibodies, the antigen-binding domain that binds to BCMA is anti-human BCMA V H It is derived from the H-Fc chimeric antibody and its humanized antibody, and the antigen-binding domain that binds to CD3 was obtained by humanizing the 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.).

[0261] The anti-BCMA / anti-CD3 multispecific antibody BC24 has three polypeptide chains, one of which (designated A10-linker-A11-Fc) contains two BCMA-binding antigen-binding domains in tandem, derived from the single variable domains 1A10-Fc and 1A11-Fc, respectively. The amino acid sequence of the polypeptide chain A10-linker-A11 is shown in SEQ ID NO:59, and the nucleotide sequence is shown in SEQ ID NO:60. The remaining two polypeptide chains of the anti-BCMA / anti-CD3 multispecific antibody BC24 are configured to contain Fab-type antigen-binding domains that bind to CD3, and these two polypeptide chains are designated 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.

[0262] The anti-BCMA / anti-CD3 multispecific antibody PC1 has two polypeptide chains, one of which (designated anti-BCMA-A10-v1-linker-A11-v1-Fc) contains two BCMA-binding antigen-binding domains in tandem, derived from the single variable domains of humanized antibodies 1A10-V1 and 1A11-V1, respectively. The N-terminus of one of the polypeptide chains in Fc is fused to an antigen-binding domain that binds BCMA (derived from 1A11-V1). The amino acid sequence of the 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:66. The other polypeptide chain of the anti-BCMA / anti-CD3 multispecific antibody PC1 (designated anti-CD3-scFv-Fc) contains an antigen-binding domain that binds CD3, in a ScFv structure containing a heavy chain variable region and a light chain variable region in tandem that bind CD3. The N-terminus of the other polypeptide chain in Fc is fused to an antigen-binding domain that binds to CD3. The amino acid sequence of the polypeptide chain anti-CD3-scFv-Fc is shown in SEQ ID NO:67, and the nucleotide sequence is shown in SEQ ID NO:68.

[0263] The anti-BCMA / anti-CD3 multispecific antibody PC2 has two polypeptide chains, one of which (designated anti-BCMA-A10-v1-Fc) contains one BCMA-binding antigen-binding domain derived from the single variable domain of humanized antibody 1A10-v1. The N-terminus of one of the polypeptide chains in Fc is fused to an antigen-binding domain that binds 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:70. The other polypeptide chain of the anti-BCMA / anti-CD3 multispecific antibody PC2 (designated anti-BCMA-A11-v1-linker-anti-CD3-scFv-Fc) contains an antigen-binding domain that binds CD3 in tandem, which is an ScFv structure containing a heavy chain variable region that binds CD3 and a light chain variable region that binds BCMA in tandem, and an antigen-binding domain that binds BCMA derived from the single variable domain of humanized antibody 1A11-v1. The N-terminus of the other polypeptide chain in Fc is fused to an antigen-binding domain that binds to CD3. The amino acid sequence of the 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.

[0264] The anti-BCMA / anti-CD3 multispecific antibody PC3 has three polypeptide chains, one of which (anti-BCMA-A11-v1-Fc) contains one BCMA-binding antigen-binding domain derived from the single variable domain of humanized antibody 1A11-v1. The N-terminus of one polypeptide chain in Fc is fused to an antigen-binding domain that binds BCMA. The amino acid sequence of the polypeptide chain anti-BCMA-A11-v1-Fc is represented by SEQ ID NO: 73, and the nucleotide sequence is represented by SEQ ID NO: 74. The remaining two polypeptide chains of the anti-BCMA / anti-CD3 multispecific antibody PC3 are configured to contain antigen-binding domains in the Fab format that bind CD3. The N-terminus of the Fab light chain is fused to an antigen-binding domain that binds BCMA derived from the single variable domain of humanized antibody 1A10-v1. The N-terminus of the other polypeptide chain in Fc is fused to an Fab heavy chain of an antigen-binding domain that binds CD3. These two polypeptide chains were named anti-CD3-HC-Fc (amino acid sequence represented by SEQ ID NO:61, nucleotide sequence represented by SEQ ID NO:62) and anti-BCMA-A10-v1-linker-anti-CD3-VL (amino acid sequence represented by SEQ ID NO:75, nucleotide sequence represented by SEQ ID NO:76), respectively.

[0265] In this example, Celgene's anti-BCMA / anti-CD3 bispecific antibody was used as a control antibody (sequence described in US20190263920A1). The anti-BCMA / anti-CD3 bispecific control antibody Benchmark (hereinafter referred to as Celgene BM or celgene BM) used in this example contains two Fab-type antigen binding domains that bind to BCMA and one Fab-type antigen binding domain that binds to CD3. One of the anti-BCMA arms has an anti-BCMA-Fab-Fc structure, and contains the polypeptide chains anti-BCMA-HC-Fc (amino acid sequence represented by SEQ ID NO: 77, nucleotide sequence represented by SEQ ID NO: 78) and anti-BCMA-LC (amino acid sequence represented by SEQ ID NO: 79, nucleotide sequence represented by SEQ ID NO: 80), and is obtained by adding H435R and Y436F mutations to anti-BCMA-HC-Fc (SEQ ID NO: 77) to increase the yield of the purification process. The structure of the other anti-BCMA / anti-CD3 arm of the anti-BCMA / anti-CD3 bispecific control antibody BM is an anti-BCMA-Fab-CD3-Fab-Fc structure and comprises the polypeptide chains anti-BCMA-VH-anti-CD3-VL-Fc (amino acid sequence shown in SEQ ID NO: 81, nucleotide sequence shown in SEQ ID NO: 82), anti-CD3-VH-CL (amino acid sequence shown in SEQ ID NO: 83, nucleotide sequence shown in SEQ ID NO: 84), and anti-BCMA-LC (amino acid sequence shown in SEQ ID NO: 79, nucleotide sequence shown in SEQ ID NO: 80).

[0266] For each anti-BCMA / anti-CD3 antibody, the DNA sequence encoding each polypeptide chain constituting the antibody was inserted into the 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. Each vector also contains an Ori sequence for autonomous replication.

[0267] To generate these antibody molecules, each antibody combination was expressed using the ExpiCHO Expression Kit (Thermo Fisher, Cat. No. A29133). First, the corresponding expression vectors were transfected at a specific ratio (transfection ratio in PC1 was 1:1.5 vector(anti-BCMA-A10-v1-linker-A11-v1-Fc):vector(anti-CD3-scFv-Fc), transfection ratio in PC2 was 1:1.5 vector(anti-BCMA-A10-v1-Fc):vector(anti-BCMA-A11-v1-linker-anti-CD3-scFv-Fc), transfection ratio in PC3 was 1:1.5:1.5 vector(anti-BCMA-A11-v1-Fc):vector(anti-CD3-HC-Fc):vector(anti-BCMA-A10-v1-linker-anti-CD3-VL), transfection ratio in PC4 was 1:1.5:1.5 vector(anti-BCMA-A11-v1-Fc):vector(anti-CD3-HC-Fc):vector(anti-BCMA-A10-v1-linker-anti-CD3-VL), transfection ratio in PC5 was 1:1.5:1.5 vector(anti-BCMA-A11-v1-Fc):vector(anti-CD3-HC-Fc):vector(anti-BCMA-A10-v1-linker-anti-CD3-VL), transfection ratio in PC6 was 1:1.5:1.5 vector(anti-BCMA-A11-v1-Fc):vector(anti-CD3-HC-Fc):vector(anti-BCMA-A10-v1-linker-anti-CD3-VL), transfection ratio in PC7 was 1:1.5:1.5 vector(anti-BCMA-A11-v1-Fc):vector(anti-CD3-HC-Fc):vector(anti- The transfection ratio of BM was 1:2:1:1.5, that is, vector (anti-BCMA-HC-Fc):vector (anti-BCMA-LC):vector (anti-BCMA-VH-anti-CD3-VL-Fc):vector (anti-CD3-VH-CL), and the transfection ratio of BC24 was 1:1.5:2, that is, vector (A10-linker-A11-Fc):vector (anti-CD3-HC-Fc):vector (anti-CD3-LC). ) was transfected into ExpiCHO cells (CHO-S, Thermo). The cells were then cultured in ExpiCHO expression medium at 37°C in an incubator with a humid atmosphere containing 8% CO2 on an orbital shaker rotating at 130 rpm. After culturing for 10 to 14 days, the cell culture supernatant was collected and the cells were removed by centrifugation. The supernatant was purified by affinity chromatography, ion exchange chromatography and gel chromatography using an AKTA purifier 100 (GE) system.

[0268] The protein concentrations of purified protein samples were determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence.

[0269] The purity of the samples collected in each tube was detected by size exclusion chromatography (SEC). Specifically, the aggregate content of the molecules was analyzed using an analytical size exclusion column, ACQUITY UPLC Protein BEH SEC column (WATERS), in a running buffer of 16 mM NaH2PO4, 34 mM Na2HPO4, 200 mM NaCl, pH 7.0 at 25°C. Based on the SEC results, samples with a purity of more than 95% were combined.

[0270] Complete molecular weight analysis was performed using a Waters LC-MS system (Waters, Singapore, USA, UK). A MAbPac RP 4 μm 2.1 × 50 mm (Thermo, USA) column was employed, with A phase (0.1% formic acid aqueous solution) and B phase (0.1% formic acid acetonitrile solution) as the mobile phase, and the measurement wavelength was 280 nm. 1 μg of protein was injected into the liquid mass spectrometer, and the gradient was 5% B to 100% B within 5.5 min. The mass spectrometer was in positive ion mode, and the scan range was 200 to 4000 m / z. Data was collected by MassLynx 4.1, and data processing was performed by UNIFI 1.8.2.169. LC-MS analysis showed that the measured molecular weight of each anti-BCMA / anti-CD3 antibody sample mentioned above was consistent with the theoretical molecular weight. Figure 18 shows the measurement results of the complete molecular weight of PC1. The main peak molecular weight of the sample was 108,516 Da, which is consistent with the theoretical molecular weight of PC1. No homologous impurity peaks were observed.

[0271] [Table 14-1]

[0272] [Table 14-2]

[0273] [Table 14-3]

[0274] [Table 14-4]

[0275] (Example 10) SPR of binding between anti-BCMA / anti-CD3 multispecific antibody and antigen The affinity of the test antibodies for human CD3e or human BCMA protein was measured using Biacore T200 (GE) as follows.

[0276] A fixed amount of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) was captured using a chip CM5 (Cytiva, catalog 29149603) coupled with anti-hIgG (Cytiva, catalog 29234600), and human CD3e (Acro, product number CDE-H5223) or human BCMA was flowed over the chip surface, and the reaction signal was detected in real time by the software Biacore to obtain binding and dissociation curves. The buffer used in the experiment was Biacore general-purpose 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). The reaction value of Anti-hIgG coupled to the surface of chip CM5 reached approximately 9000 RU, and approximately 200 RU of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) were captured. After that, the signal values ​​of the interaction between each concentration of human CD3e protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) or human BCMA (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) and anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) were measured. The flow rate of the flow cell was 50 μl / min, the binding time was 240 s, the dissociation time was 1400 s, the regeneration with 3M MgCl2 (GE) was 60 s, and the baseline was stable. The results were calculated using the affinity and kinetics 1:1 binding model of the biacore evaluation software. The affinity of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, Celgene BM) to human CD3e protein or human BCMA is shown in Table 10.

[0277] [Table 15]

[0278] Example 11 Binding of anti-BCMA / anti-CD3 multispecific antibodies to BCMA on multiple myeloma cells Flow cytometry was used to analyze the binding of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) to BCMA on BCMA-expressing NCI-H929 cells (a BCMA-high expressing cell line, obtained from Nanjing Kebai Biological), MM1S cells (a BCMA-moderate expressing cell line, obtained from Beina Biological), and RPMI-8226 cells (a BCMA-low expressing cell line, obtained from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). Trastuzumab was used as a negative control.

[0279] The detailed experimental procedure is as follows.

[0280] Logarithmically growing NCI-H929 cells (BCMA+++), MM1S cells (BCMA++), and RPMI-8226 cells (BCMA+) were cultured at a cell density of 5 × 10 in RPMI medium (Hyclon, catalog number SH30809.01) containing 2% FBS (fetal bovine serum). 6 ~10×10 6The antibody was adjusted to cells / ml and seeded at 50 μl per well in a 96-well U-shaped cell culture plate (Costar, Catalog No.: 3799). Various concentrations of the test antibody were prepared using the above medium. For BCMA high and medium expressing cells, the antibody concentration was up to 440 nM, and diluted 5-fold to a total of 9 concentration steps, and for BCMA low expressing cells, the antibody concentration was up to 2200 nM, and diluted 5-fold to a total of 10 concentration steps. 50 μl of each antibody was added per well to the 96-well cell culture plate, mixed well, and incubated at 4°C for 1 hour. After washing the cells with pre-chilled running buffer (MACS, Cat. No. 130-091-221), the supernatant was discarded, and 100 μl of pre-chilled fluorescently labeled sheep anti-human IgG antibody (Jackson, Cat. No. 109-116-170) was added per well to resuspend the cells, mixed well, and incubated at 4° C. for 30 minutes. After washing, 100 μl of pre-chilled running buffer was added to resuspend the cells, mixed well, and flow collection was performed using the Attune TM NxT flow cytometer, and data analysis was performed using the Graphpad Prism5 software.

[0281] Figures 7-9 and Table 11 show the binding ability of anti-BCMA / anti-CD3 multispecific antibodies to NCI-H929 cells (BCMA+++), MM1S cells (BCMA++) and RPMI-8226 cells (BCMA+). The results show that in target cells with high to moderate BCMA expression, PC1, PC2, PC3 and BC24 all exhibited superior maximum binding to the target antigen than Celgene BM. In target cells with low BCMA expression, PC3 and BC24 exhibited superior binding to the target antigen than the other samples.

[0282] [Table 16]

[0283] Example 12: Binding of anti-BCMA / anti-CD3 multispecific antibodies to CD3 on T cells Binding of anti-BCMA / anti-CD3 antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) to CD3 on the T cell surface was analyzed by flow cytometry. Trastuzumab was used as a negative control.

[0284] The detailed experimental procedure is as follows.

[0285] Human PBMC cells were purified by CD3 magnetic beads (Miltenyi, Cat. No. 130-097-043) as follows: + T cell isolation was performed. Cryopreserved PBMCs were lysed, washed twice by centrifugation, and counted. 7 Add running buffer (MACS, Cat. No. 130-091-221) at a ratio of 80 μl per cell and 10 7 20 μl of CD3 magnetic beads were added per cell, mixed well, and incubated at 4° C. for 15 minutes. 7 The cells were washed by adding 1-2 ml of buffer per cell, centrifuged, the supernatant discarded, and the cell pellet was resuspended by adding 500 μl of buffer. The LS separation column (Miltenyi, Cat. No. 130-042-401) was placed on the MidiMACS Starting Kit (LS) magnetic stand (Miltenyi, Cat. No. 130-091-051), rinsed, and the cell suspension was added and washed three times. The LS separation column was removed from the magnetic stand, placed in a clean centrifuge tube, and 5 ml of buffer was added to separate the CD3 + T cells were collected and counted. CD3 + T cells at a cell density of 5 × 10 6 ~10×10 6The cells were adjusted to 1000 cells / ml and seeded in a 96-well U-shaped cell culture plate (Costar, Catalog No.: 3799) at 50 μl per well. Various concentrations of the test antibodies were prepared using the above medium, with the antibody concentration up to 2200 nM, and diluted 5-fold to a total of 10 concentration steps. 50 μl of each antibody concentration was added per well to the above 96-well cell culture plate, mixed well, and incubated at 4°C for 1 hour. After washing the cells with pre-cooled running buffer (MACS, Catalog No. 130-091-221), the supernatant was discarded, and pre-cooled fluorescently labeled sheep anti-human IgG antibody (Jackson, Catalog No. 109-116-170) was added per well at 100 μl, resuspended, mixed well, and incubated at 4°C for 30 minutes. After washing, 100 μl of pre-chilled running buffer was added to resuspend the cells, and the cells were mixed well. Flow collection was then performed using an Attune™ NxT flow cytometer, and data analysis was performed using the Graphpad Prism5 software.

[0286] Figure 10 and Table 12 show the CD3 + The binding ability of anti-BCMA / anti-CD3 multispecific antibodies to T cells is shown. The results show that each anti-BCMA / anti-CD3 multispecific antibody binds to CD3 + It was able to bind to T cells.

[0287] [Table 17]

[0288] Example 13: Killing of multiple myeloma target cells NCI-H929 by anti-BCMA / anti-CD3 multispecific antibodies Human PBMCs (peripheral blood mononuclear cells) were used to administer T cells, and the killing effect of antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) on target cells (NCI-H929 BCMA+++, obtained from Nanjing Kebai Biology) was studied. Trastuzumab was used as a negative control.

[0289] The detailed experimental procedure is as follows.

[0290] Target cells: NCI-H929 cells. Culture the cells at a cell density of 5 × 10 using 1640 experimental medium containing 2% FBS (fetal bovine serum). 5 The antibody was adjusted to 1000 cells / ml and seeded at 50 μl per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Various concentrations of the test antibody were prepared using experimental medium, with the antibody concentration at the maximum of 8 nM, and diluted 5-fold to a total of 10 concentration steps. Each antibody concentration was added at 50 μl per well to the 96-well cell culture plate. Human PBMCs, the effector cells, were cultured at a cell density of 2.5 × 10 6 The cells / ml were adjusted and 100 μl was added per well. The following groups were prepared: treatment group (50 μl of target cells + 100 μl of effector cells + 50 μl of antibody), target cell group (50 μl of target cells + 150 μl of medium), effector cell group (100 μl of human PBMC + 100 μl of medium), target cell + effector cell group (50 μl of target cells + 100 μl of effector cells + 50 μl of medium), blank control group (200 μl of medium), lysis solution control group (200 μl of medium + lysis solution 20 μl), and maximum target cell release group (50 μl of target cells + 150 μl of medium + lysis solution 20 μl). The effector cells and target cells were co-cultured for 24 hours at a ratio of 10:1. 45 minutes before measurement, lysis solution (Promega, Catalog No. G182A) was added to the target cell maximum release group and lysis solution control group at 20 μl / well. CytoTox96® nonradioactive cytotoxicity assay kit (cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780) was used to measure the cell lysis rate.

[0291] Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 )×100% Figure 11 and Table 13 show the lysis rate of NCI-H929 / BCMA+++ tumor cells by effector cells induced by anti-BCMA / anti-CD3 multispecific antibodies. The anti-BCMA / anti-CD3 antibody PC1 induced a better cell killing effect than PC2 and PC3. The EC50 of PC1 was about 0.067nM, and the EC50 of PC2 and PC3 were about 0.53nM and 0.19nM, respectively. BC24 and Celgene BM had a better killing effect on NCI-H929 than PC1, PC2, and PC3. The EC50 of BC24 was about 0.022nM, and the EC50 of Celgene BM was about 0.044nM.

[0292] [Table 18]

[0293] (Example 14) Killing of multiple myeloma target cells MM1S by anti-BCMA / anti-CD3 multispecific antibodies Human PBMCs (peripheral blood mononuclear cells) were used to administer T cells, and the killing effect of antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) on target cells (MM1S BCMA++, obtained from Kitano Bio) was studied. Trastuzumab was used as a negative control.

[0294] The detailed experimental procedure is as follows.

[0295] The target cells, MM1S cells, were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) at a cell density of 5 × 10 5 The antibody was adjusted to 1000 cells / ml and seeded at 50 μl per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Various concentrations of the test antibody were prepared using experimental medium, with the antibody concentration at the maximum of 8 nM, and diluted 5-fold to a total of 10 concentration steps. Each antibody concentration was added at 50 μl per well to the 96-well cell culture plate. Human PBMCs, the effector cells, were cultured at a cell density of 2.5 × 10 6The cells / ml were adjusted and 100 μl was added per well. The following groups were prepared: treatment group (50 μl of target cells + 100 μl of effector cells + 50 μl of antibody), target cell group (50 μl of target cells + 150 μl of medium), effector cell group (100 μl of human PBMC + 100 μl of medium), target cell + effector cell group (50 μl of target cells + 100 μl of effector cells + 50 μl of medium), blank control group (200 μl of medium), lysis solution control group (200 μl of medium + lysis solution 20 μl), and maximum target cell release group (50 μl of target cells + 150 μl of medium + lysis solution 20 μl). The effector cells and target cells were co-cultured for 24 hours at a ratio of 10:1. 45 minutes before measurement, lysis solution (Promega, Catalog No. G182A) was added to the target cell maximum release group and lysis solution control group at 20 μl / well. CytoTox96® nonradioactive cytotoxicity assay kit (cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780) was used to measure the cell lysis rate.

[0296] Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 )×100% Figure 12, Table 14 show the lysis rate of MM1S / BCMA++ tumor cells by effector cells induced by anti-BCMA / anti-CD3 multispecific antibodies. Anti-BCMA / anti-CD3 multispecific antibody PC1 induced better cell killing than PC2 and PC3. The EC50 of PC1 was about 0.0084nM, and the EC50 of PC2 and PC3 were about 0.061nM and 0.041nM, respectively. PC3 showed a better maximum lysis rate. Celgene BM had the weakest killing effect on MM1S, with an EC50 of about 0.085nM and a maximum lysis rate of 23%.

[0297] [Table 19]

[0298] Example 15: Killing of multiple myeloma target cells RPMI-8226 by anti-BCMA / anti-CD3 multispecific antibodies Human PBMCs (peripheral blood mononuclear cells) were used to administer T cells, and the killing effect of antibodies (including BC24, PC1, PC2, PC3, and Celgene BM) on target cells (RPMI-8226 BCMA+, obtained from the Center for Basic Medical Cells, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) was studied. Trastuzumab was used as a negative control.

[0299] The detailed experimental procedure is as follows.

[0300] The target cells, RPMI-8226 cells, were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) at a cell density of 5 × 10 5 The antibody was adjusted to 1000 cells / ml and seeded at 50 μl per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Various concentrations of the test antibody were prepared using experimental medium, with the antibody concentration at the maximum of 8 nM, and diluted 5-fold to a total of 10 concentration steps. Each antibody concentration was added at 50 μl per well to the 96-well cell culture plate. Human PBMCs, the effector cells, were cultured at a cell density of 2.5 × 10 6The cells / ml were adjusted and 100 μl was added per well. The following groups were prepared: treatment group (50 μl of target cells + 100 μl of effector cells + 50 μl of antibody), target cell group (50 μl of target cells + 150 μl of medium), effector cell group (100 μl of human PBMC + 100 μl of medium), target cell + effector cell group (50 μl of target cells + 100 μl of effector cells + 50 μl of medium), blank control group (200 μl of medium), lysis solution control group (200 μl of medium + lysis solution 20 μl), and maximum target cell release group (50 μl of target cells + 150 μl of medium + lysis solution 20 μl). The effector cells and target cells were co-cultured for 24 hours at a ratio of 10:1. 45 minutes before measurement, lysis solution (Promega, Catalog No. G182A) was added to the target cell maximum release group and lysis solution control group at 20 μl / well. CytoTox96® nonradioactive cytotoxicity assay kit (cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780) was used to measure the cell lysis rate.

[0301] Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 )×100% Figure 13 and Table 15 show the lysis rate of RPMI-8226 / BCMA+ tumor cells by effector cells induced by anti-BCMA / anti-CD3 multispecific antibodies. The anti-BCMA / anti-CD3 multispecific antibody PC1 showed a similar maximum lysis rate against RPMI-8226 but a lower EC50 compared to BC24. PC2 and PC3 showed similar killing effects against RPMI-8226, with EC50s of 0.0185nM and 0.0142nM for PC2 and PC3, respectively, and both had maximum lysis rates of 39%. Celgene BM showed the weakest killing effect against RPMI-8226, with an EC50 of 0.27nM and a maximum lysis rate of 29%.

[0302] [Table 20]

[0303] Example 16: T cell activation induced by anti-BCMA / anti-CD3 multispecific antibodies CD69 and CD25 are T cell activation markers. This example relates to the analysis of T cell activation by multispecific antibodies (including BC24, PC1, PC2, PC3, Celgene BM) in the presence of myeloma target cells. Trastuzumab was used as a negative control (NC).

[0304] The detailed experimental procedure is as follows.

[0305] The target cells, NCI-H929 cells and MM1S, were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) at a cell density of 5 × 10 5 The antibody was adjusted to 1000 cells / ml and seeded at 50 μl per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Various concentrations of the test antibody were prepared using experimental medium, with the antibody concentration at the maximum of 8 nM, and diluted 5-fold to a total of 10 concentration steps. Each antibody concentration was added at 50 μl per well to the 96-well cell culture plate. Human PBMCs, the effector cells, were cultured at a cell density of 2.5 × 10 6The cells / ml were adjusted and 100 μl was added per well. There were treatment group (50 μl of target cells + 100 μl of effector cells + 50 μl of antibody), non-treatment group (50 ul of medium + 100 μl of effector cells + 50 μl of antibody), target cell group (50 μl of target cells + 150 μl of medium), effector cell group (100 μl of human PBMC + 100 μl of medium), target cell + effector cell group (50 μl of target cells + 100 μl of effector cells + 50 μl of medium), and blank control group (200 μl of medium). Trastuzumab was used as a negative control antibody NC, and the effector cells and target cells were co-cultured at a ratio of 10:1 for 24 hours. The cells were centrifuged to remove the supernatant, washed with pre-chilled running buffer (MACS, Cat. No. 130-091-221), and after removing the supernatant, 100 μl of running buffer was added per well, the cells were mixed well, and CD4-BV421 (BD, Cat. No. 564713), CD8-PE-Cy7 (BD, Cat. No. 557746), CD25-APC (BD, Cat. No. 555434), and CD69-FITC (BD, Cat. No. 555530) were added at 2.5 μl per well and incubated on ice for 30 minutes. The cells were washed with pre-chilled running buffer, and 75 μl / well of running buffer was added to resuspend the cells, mixed well, and analyzed by flow cytometer.

[0306] 14 and 15 show the degree of T cell activation in the presence and absence of target cells NCI-H929 or MM1S when the concentration of anti-BCMA / anti-CD3 antibody was 8 nM, with the vertical axis representing the percentage of CD8 or CD4 cells positive for activation markers CD25 or CD69, and the horizontal axis representing groups. In the figures, CD4+CD25+ / CD4+ represents the percentage of CD4 cells positive for CD25 among the CD4 cells of a certain group, CD4+CD69+ / CD4+ represents the percentage of CD4 cells positive for CD69 among the CD4 cells of a certain group, CD8+CD25+ / CD8+ represents the percentage of CD8 cells positive for CD25 among the CD8 cells of a certain group, and CD8+CD69+ / CD8+ represents the percentage of CD8 cells positive for CD69 among the CD8 cells of a certain group. The results showed that in the absence of target cells, T cells were barely activated, but in the presence of target cells, antibodies BC24, PC1, PC2, and PC3 were able to activate T cells.

[0307] Example 17: Cytokine release induced by anti-BCMA / anti-CD3 multispecific antibodies The release of cytokines induced by the multispecific antibodies in the presence of target cells was studied by measuring the contents of IL-2, IL-6, TNF-α and IFN-γ. The safety of the multispecific antibodies was evaluated by the amount of cytokine release.

[0308] The detailed experimental procedure is as follows.

[0309] The target cells, NCI-H929 cells, were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) at a cell density of 5 × 10 5The antibody was adjusted to 1000 cells / ml and seeded at 50 μl per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Various concentrations of the test antibody were prepared using experimental medium, with the antibody concentration at the maximum of 8 nM, and diluted 5-fold to a total of 10 concentration steps. Each antibody concentration was added at 50 μl per well to the 96-well cell culture plate. Human PBMCs, the effector cells, were cultured at a cell density of 2.5 × 10 6 The cells / ml were adjusted and 100 μl was added per well. Standard group, treatment group (50 μl of target cells + 100 μl of effector cells + 50 μl of antibody), target cell group (50 μl of target cells + 150 μl of medium), effector cell group (100 μl of human PBMC + 100 μl of medium), target cell + effector cell group (50 μl of target cells + 100 μl of effector cells + 50 μl of medium), effector cell + antibody group (100 μl of effector cells + 50 μl of antibody + 50 μl of medium), and blank control group (200 μl of medium) were prepared, and the effector cells and target cells were co-cultured at a ratio of 10:1 for 24 hours. After centrifugation, the supernatant was collected and the samples were treated using the human-IL2 kit (Cisbio, catalog number 62HIL02PEG), human-IL6 kit (Cisbio, catalog number 62HIL06PEG), human-TNF-α kit (Cisbio, catalog number 62HTNFAPEG), or human-IFN-γ kit (Cisbio, catalog number 62HIFNGPEG) assay kits according to the kit instructions, and measurement analysis was performed using a microplate reader (Molecular Devices, product number Paradigm).

[0310] The Ratio values ​​for all samples were calculated, and the Standard Ratio (Net) was plotted on the Y-axis and Log (Standard concentration value) on the X-axis. Four-parameter fitting was performed using Graph Pad Prism, and the Ratio (Net) values ​​of the standard and test products were substituted to obtain the log (measured concentration), and the concentration measurement values ​​of each cytokine in the test products were calculated.

[0311] Figure 16, Table 16 shows the amount of cytokines IL-2, IL-6, TNF-α and IFN-γ released after 24 hours of co-culture of NCI-H929 cells and effector cells with various concentrations of anti-BCMA / anti-CD3 antibodies. Each tested antibody had low release of IL-2. The BC24, PC1, PC2 and PC3 multispecific antibodies of the present invention had lower release levels of cytokines IL-6, TNF-α and IFN-γ than Celgene BM, and showed better safety.

[0312] [Table 21]

[0313] Example 18: Anti-BCMA / anti-CD3 dual antibody inhibits proliferation of multiple myeloma target cells NCI-H929 in a mouse tumor model The antitumor activity of anti-BCMA / anti-CD3 multispecific antibodies was analyzed using a mouse model of human myeloma NCI-H929 cells. 6 0.5×10 human peripheral lymphocytes (hPBMC) 6 The two were mixed so that the number of mice was 100–300 mm and then inoculated into the right axilla of 8-10 week-old female B-NDG mice (obtained from Biocytogen, Inc., Certificate No.: 320726200100179034) under sterile conditions. 3 When the mice reached 10 days after the experiment, they were randomly divided into two groups: (1) model group (blank control group, saline) and (2) PC1 group (test antibody PC1). On day 0, the mice were divided into groups and administered twice a week for one week at a dose of 1.5mpk and a volume of 10ml / kg. Body weight and tumor diameter were measured every three days, and the condition of the animals was observed every day. Observation was discontinued on day 17. During the experiment, tumor volumes were measured on days 0, 4, 8, 11, 14, and 17. Tumor volumes and tumor inhibition rates were calculated using the following formulas.

[0314] Tumor volume (TV) = (length x width 2 ) / 2 Relative tumor volume (RTV) = TV t / TV0 (Wherein, TV0 is the tumor volume at the time of grouping, TV t is the tumor volume at each measurement.)

[0315] Relative tumor growth rate T / C(%)=(T RTV / C RTV )×100% (In the formula, T RTV is the RTV of the treatment group, and C RTV is the RTV of the blank control group.)

[0316] Tumor inhibition rate (TGI)=(1-T / C)×100%.

[0317] The experimental results are shown in Figure 17. Compared with the model group, the PC1 antibody administration group significantly inhibited the growth of human multiple myeloma NCI-H929 subcutaneously transplanted tumors. 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.

[0318] Example 19 Pharmacokinetics of a single intravenous injection of anti-BCMA / anti-CD3 multispecific antibodies in cynomolgus monkeys Cynomolgus monkeys (numbers 1M01-1M03, 2M01-2M03, 3 per dose group, male, body weight: 2-3 kg) were intravenously injected with PC1 injection solution once. The dose of PC1 polyspecific antibody was 1 or 9 mg / kg, and the intravenous infusion time was 30 minutes. Blood samples were taken before and after administration, and within 1 hour, the blood samples were centrifuged at 4000 rpm for about 10 minutes (2-8°C) to isolate serum. Drug concentrations in the blood samples at each time point were measured by ELISA. Sampling was performed at 0 hour (before administration), and 0.5, 4, 24, 48, 96, 168, 240, and 336 hours after completion of administration.

[0319] The parameters are as follows:

[0320] C max : Maximum observed drug concentration AUC: Area under the plasma concentration-time curve T 1 / 2 :Half-life Cl: Clearance rate MRT: Drug retention time According to the experimental results, the anti-BCMA / anti-CD3 multispecific antibody was intravenously administered once at 1 mg / kg and 9 mg / kg, and the terminal elimination half-life of the drug, t 1 / 2 are 105 hours and 112 hours, respectively, and C max were 19 and 200 μg / mL, respectively, and AUC last The serum exposure to the drug was found to increase essentially in proportion to the dose.

[0321] [Table 22]

Claims

1. (i) a first antigen-binding portion that binds to a first antigen; and (ii) a second antigen-binding portion that binds to a second antigen; and (iii) a third antigen-binding moiety that binds to the first antigen, the first antigen is BCMA and the second antigen is a T cell activation antigen, and the first antigen-binding moiety and the third antigen-binding moiety are both single variable domains, each independently comprising: (b) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (c) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15; (a) a CDR1 comprising the amino acid sequence represented by SEQ ID NO:7, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:8, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:9; or (d) A multispecific antibody comprising any one of CDR1 comprising the amino acid sequence represented by SEQ ID NO: 16, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:

18.

2. The first antigen-binding portion and the third antigen-binding portion each independently comprise: (b) a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; or (c) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO:

15. The multispecific antibody of claim 1, comprising any one of the following:

3. The multispecific antibody of claim 2, wherein the first antigen-binding portion comprises, as complementarity determining regions, a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and the third antigen-binding portion comprises, as complementarity determining regions, a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:

15.

4. The multispecific antibody of claim 2, wherein the third antigen-binding portion comprises, as complementarity determining regions, a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12, and the first antigen-binding portion comprises, as complementarity determining regions, a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:

15.

5. or the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence represented by SEQ ID NO:21, 37 or 38, and the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence represented by SEQ ID NO:23, 39 or 40; or 2. The multispecific antibody of claim 1, wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 37, and the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO:

39.

6. or the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence represented by SEQ ID NO: 23, 39 or 40, and the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence represented by SEQ ID NO: 21, 37 or 38; or 2. The multispecific antibody of claim 1, wherein the first antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO: 39, and the third antigen-binding portion comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence represented by SEQ ID NO:

37.

7. The first antigen-binding portion and the third antigen-binding portion are (1) a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO:21 and a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO:23, 39, or 40; (2) a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 37 and a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (3) a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 38 and a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (4) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO:21 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO:23, 39, or 40; (5) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 37, and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (6) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 38 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 23, 39, or 40; (7) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 41 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 42; (8) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 42 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 41; (9) a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 37 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 39; or (10) The multispecific antibody of claim 1, which is any one selected from a third antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO: 39 and a first antigen-binding portion comprising the amino acid sequence represented by SEQ ID NO:

37.

8. The multispecific antibody of claim 1 , wherein the T cell activation antigen is CD3.

9. the second antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising, as complementarity determining regions, an HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 43, an HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and an HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, and the light chain variable region comprising an LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 48; or 2. The multispecific antibody of claim 1 , wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the variable region represented by SEQ ID NO: 49, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region represented by SEQ ID NO:

50.

10. 10. The multispecific antibody of claim 9, wherein the second antigen-binding portion comprises a heavy chain variable region having an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 49 and a light chain variable region having an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 50; or wherein the heavy chain variable region of the second antigen-binding portion comprises the amino acid sequence set forth in SEQ ID NO: 49 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

50.

11. (iv) a Fc region composed of two Fc polypeptides.

12. (1) the second antigen-binding portion is an ScFv, and the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused at its C-terminus to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion; (2) the second antigen-binding portion is an ScFv, and the first antigen-binding portion is fused C-terminally to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused C-terminally to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused C-terminally to the N-terminus of the second antigen-binding portion; (3) the second antigen-binding portion is a Fab, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused at the C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the first antigen-binding portion; or (4) The multispecific antibody of claim 11, wherein the second antigen-binding portion is a Fab, the first antigen-binding portion is fused at its C-terminus to the N-terminus of one Fc polypeptide in the Fc region, the second antigen-binding portion is fused at its C-terminus of a Fab heavy chain to the N-terminus of the other Fc polypeptide in the Fc region, and the third antigen-binding portion is fused at its C-terminus to the N-terminus of the Fab light chain of the second antigen-binding portion or the N-terminus of the Fab heavy chain of the second antigen-binding portion.

13. the Fc region is an IgG Fc region, a human IgG Fc region, a human IgG1 or a human IgG4 Fc region; Optionally, the Fc Region may comprise amino acid substitutions that facilitate association of the two Fc polypeptides of the Fc Region; and / or the Fc region may comprise amino acid substitutions that reduce the binding affinity of the Fc region to an Fc receptor and / or an effector function; or / and The multispecific antibody of claim 11, wherein the Fc region optionally comprises an amino acid substitution that reduces or eliminates binding of the CH3 region of one Fc polypeptide in the Fc region to Protein A.

14. (1) A polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 65, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 67; (2) A polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO:69, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO:71; (3) A polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO:59, a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO:61, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO:63; (4) A polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 73, a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 61, and a polypeptide chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence represented by SEQ ID NO: 75; (5) A polypeptide chain comprising two polypeptide chains, one comprising the amino acid sequence represented by SEQ ID NO:65 and the other comprising the amino acid sequence represented by SEQ ID NO:67; (6) A polypeptide chain comprising the amino acid sequence represented by SEQ ID NO:69 and a polypeptide chain comprising the amino acid sequence represented by SEQ ID NO:71; (7) A polypeptide chain comprising an amino acid sequence represented by SEQ ID NO:59, a polypeptide chain comprising an amino acid sequence represented by SEQ ID NO:61, and a polypeptide chain comprising an amino acid sequence represented by SEQ ID NO:63; or (8) The multispecific antibody according to claim 1, which is composed of three polypeptide chains: a polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 73, a polypeptide chain comprising the amino acid sequence represented by SEQ ID NO: 61, and a polypeptide chain comprising the amino acid sequence represented by SEQ ID NO:

75.

15. An isolated nucleic acid comprising a polynucleotide encoding a multispecific antibody according to any one of claims 1 to 14.

16. A vector comprising the nucleic acid described in claim 15.

17. A host cell comprising the nucleic acid described in claim 15.

18. A method for producing a multispecific antibody, comprising culturing the host cell described in claim 17 to express the multispecific antibody described in any one of claims 1 to 14, and isolating and purifying the multispecific antibody from the system.

19. A pharmaceutical composition comprising the multispecific antibody of claim 1 and a pharma- ceutical acceptable carrier.

20. 20. Use of a multispecific antibody according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 19 for the manufacture of a pharmaceutical composition or a medicament for treating a disease associated with BCMA expression in a subject, said disease being lymphoma, multiple myeloma, leukemia, systemic lupus erythematosus or rheumatoid arthritis.

21. 1. A BCMA-binding antibody comprising a single variable domain, said single variable domain comprising: (2) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 11, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 12; (3) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 13, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 14, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 15; (1) a CDR1 comprising the amino acid sequence represented by SEQ ID NO:7, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:8, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:9; or (4) A BCMA-binding antibody comprising a CDR1 having an amino acid sequence represented by SEQ ID NO: 16, a CDR2 having an amino acid sequence represented by SEQ ID NO: 17, and a CDR3 having an amino acid sequence represented by SEQ ID NO: 18.

Images