Anti-BCMA nanobody and its applications

Abstract

Antibodies or antigen-binding fragments thereof that specifically bind to BCMA, their encoding nucleic acids, expression vectors and expressing cells, methods of manufacture, pharmaceutical compositions, and uses to treat disease, e.g., in the treatment of tumors.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to a Chinese patent application filed on December 03, 2021 with the State Intellectual Property Office of China, bearing patent application number 202111468274.X and titled "BCMA nanobody and its application", and a Chinese patent application filed on August 19, 2022 with the State Intellectual Property Office of China, bearing patent application number 20221100054.9 and titled "Anti-BCMA nanobody and its application". The above prior applications are incorporated herein by reference in their entirety.

[0002] The present application relates to the biomedicine field, specifically to anti-BCMA nanobodies and their applications. [Background technology]

[0003] Multiple myeloma (MM) is a malignant plasma cell tumor that originates from the bone marrow and is a type of B-cell lymphoma, also called plasmacytoma. It is characterized by abnormal proliferation of bone marrow plasma cells accompanied by overproduction of monoclonal immunoglobulin or light chain (M protein), and a very small number of patients may have non-secretory MM that does not produce M protein. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, and kidney damage. Because normal immunoglobulin production is suppressed, various bacterial infections are likely to occur.

[0004] The incidence of multiple myeloma accounts for 1% of all tumors and 10-15% of hematological malignancies. The male-female ratio is 1.6:1, and the majority of patients are >40 years old. Treatment of multiple myeloma includes chemotherapy and hematopoietic stem cell transplantation. Here, immunomodulators such as lenalidomide and protease inhibitors such as bortezomib have shown good efficacy as single agents or in combination, and have become common treatments for multiple myeloma patients. However, multiple myeloma is still considered an incurable disease, and current treatments only alleviate the symptoms of multiple myeloma, none of which can completely remove the tumor, and most patients end up relapsing. Therefore, there is a strong demand for new treatments.

[0005] B cell maturation antigen (BCMA), also known as CD269 or TNFRSF17, is a member of the tumor necrosis factor receptor superfamily and was first discovered in the early 1990s. The receptor is mainly expressed on the surface of mature B lymphocytes and plasma cells, and is a marker protein for B lymphocyte maturation, and is rarely expressed on other tissue cells. Structurally, BCMA consists of three main domains: an extracellular segment (aa1-54), a transmembrane region (aa55-77), and an intracellular segment (aa78-184). B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) are the main ligands of BCMA, which transmit cell stimulatory signals by interacting with BCMA, activating the TRAF-dependent NF-κB, JNK pathway, and increasing the proliferation and survival of B cells. BCMA is highly expressed in MM cells and is an ideal antigen target for multiple myeloma. BCMA has a human-monkey homology of 88%, and screening for cross-binding antibodies poses certain difficulties.

[0006] Although therapeutic approaches such as CAR T, bispecific antibodies, and ADCs against BCMA have made important progress and shown promising prospects, there is still an urgent need for the development of more efficient next-generation BCMA-specific antibodies and biotherapeutic products based on them. Summary of the Invention

[0007] The present application provides antibodies or antigen-binding fragments thereof that specifically bind to BCMA, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors, host cells, pharmaceutical compositions, kits, methods of manufacture and their applications in the treatment of disease and detection of BCMA.

[0008] In one aspect, the application provides an antibody or antigen-binding fragment thereof that specifically binds to BCMA, wherein the antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises an HCDR1 of a VHH domain as shown in any one of SEQ ID NOs:7-21, 24-31 and 92-101, the CDR2 comprises an HCDR2 of a VHH domain as shown in any one of SEQ ID NOs:7-21, 24-31 and 92-101, and the CDR3 comprises an HCDR3 of a VHH domain as shown in any one of SEQ ID NOs:7-21, 24-31 and 92-101.

[0009] In another aspect, the present application provides a multispecific antigen binding molecule, wherein said multispecific antigen binding molecule comprises an antibody or antigen binding fragment thereof and an antigen binding molecule that binds to another antigen other than BCMA, or that binds to a different BCMA epitope than the antibody or antigen binding fragment thereof.

[0010] In another aspect, the present application provides an isolated nucleic acid fragment, wherein said nucleic acid fragment encodes the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned multispecific antigen-binding molecule.

[0011] In another aspect, the present application provides a vector, wherein said vector comprises the nucleic acid fragment described above.

[0012] In another aspect, the present application provides a host cell, wherein said host cell comprises the nucleic acid fragment or the vector.

[0013] In another aspect, the present application provides a method of producing said antibody or antigen-binding fragment thereof, or said multispecific antigen-binding molecule, said method comprising culturing said cell and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by said cell.

[0014] In another aspect, the present application provides a pharmaceutical composition, wherein said pharmaceutical composition comprises said antibody or antigen-binding fragment thereof, said multispecific antigen-binding molecule, said nucleic acid fragment, said vector, or a product produced according to said method.

[0015] In another aspect, the present application provides a method of treating a tumor or cancer, wherein the method comprises administering to a subject an effective amount of the above-described antibody or antigen-binding fragment thereof, the above-described multispecific antigen-binding molecule, the above-described nucleic acid fragment, the above-described vector, a product produced according to the above-described method, or the above-described pharmaceutical composition.

[0016] In another aspect, the present application provides the use of said antibody or antigen-binding fragment thereof, said multispecific antigen-binding molecule, said nucleic acid fragment, said vector, said product produced according to said method, or said pharmaceutical composition in the manufacture of a medicament for treating tumors or cancer.

[0017] In another aspect, the present application provides a use of said antibody or antigen-binding fragment thereof, said multispecific antigen-binding molecule, said nucleic acid fragment, said vector, a product produced according to said method, or said pharmaceutical composition for treating tumor or cancer.

[0018] In another aspect, the present application provides a kit, wherein said kit comprises the above-described antibody or antigen-binding fragment thereof, the above-described multispecific antigen-binding molecule, the above-described nucleic acid fragment, the above-described vector, a product produced according to the above-described method, or the above-described pharmaceutical composition.

[0019] In another aspect, the application provides a method of detecting BCMA expression in a biological sample, said method comprising contacting said biological sample with said antibody or antigen binding fragment thereof under conditions allowing the formation of a complex between said antibody or antigen binding fragment thereof and BCMA.

[0020] In another aspect, the present application provides the use of the aforementioned antibody or antigen-binding fragment thereof in the manufacture of a BCMA detection reagent.

[0021] The present application provides an antibody or antigen-binding fragment thereof with high affinity for the BCMA target, and can better block the binding of BCMA to its ligand APRIL, thereby providing a better option for BCMA antibody drugs and cell therapy products, and is of great significance in filling the shortage of treatment options for multiple myeloma, especially relapsed / refractory multiple myeloma. [Brief description of the drawings]

[0022] [Figure 1A] FIG. 1 shows the binding reaction between a control antibody and human BCMA-His protein detected by ELISA. [Figure 1B] FIG. 1 shows the binding reaction between a control antibody and monkey BCMA-His protein detected by ELISA. [Figure 2A] FIG. 1 shows the FACS results of detecting the BCMA expression level in H929 cells using REGN5459-hIgG1 and HPN217-hHcAb antibodies. [Figure 2B] FIG. 1 shows the FACS results of detecting the BCMA expression level in U266 cells using REGN5459-hIgG1 and HPN217-hHcAb antibodies. [Diagram 3] FIG. 1 shows the FACS results of detecting the BCMA expression level in Flp-inCHO-human BCMA cells using REGN5459-hIgG1 antibody. [Figure 4] FIG. 1 shows the FACS results of detecting the BCMA expression level in Flp-inCHO-monkey BCMA cells using REGN5459-hIgG1 antibody. [Diagram 5]FIG. 1 shows the binding reaction of recombinant candidate antibodies with human BCMA-his protein detected by ELISA. [Figure 6] FIG. 1 shows the binding reaction of recombinant candidate antibodies with monkey BCMA-his protein detected by ELISA. [Figure 7] FIG. 1 shows the binding reaction of recombinant candidate antibodies with H929 tumor cells detected by FACS. [Figure 8] FIG. 1 shows the binding reaction of recombinant candidate antibodies with U266 tumor cells detected by FACS. [Figure 9] FIG. 1 shows the blocking effect of recombinant candidate antibodies on the binding of the ligand APRIL to human BCMA protein detected by competitive ligand binding ELISA. [Figure 10] FIG. 1 shows the detection of the binding reaction of candidate humanized Nanobodies with human BCMA-his protein by ELISA. [Figure 11] FIG. 1 shows the detection of the binding reaction of candidate humanized nanobodies with monkey BCMA-his protein by ELISA. [Figure 12] FIG. 1 shows the detection of the binding reaction of candidate humanized nanobodies with H929 tumor cells by FACS. [Figure 13] FIG. 1 shows the binding reaction between humanized candidate nanobodies and U266 tumor cells detected by FACS. [Figure 14] FIG. 13: Blocking effect of humanized candidate Nanobodies on the binding of the ligand APRIL to human BCMA protein detected by competitive ligand binding ELISA. [Figure 15] FIG. 1 shows the binding reaction between a chimeric antibody and human BCMA-his protein detected by ELISA. [Figure 16A] FIG. 1 shows the binding reaction between a chimeric antibody and H929 tumor cells detected by FACS. [Figure 16B] FIG. 1 shows the binding reaction between a chimeric antibody and U266 tumor cells detected by FACS. [Figure 17]FIG. 1 shows the blocking effect of chimeric antibodies on the binding of the ligand APRIL to human BCMA protein detected by competitive ligand binding ELISA. Detailed Description of the Invention

[0023] Definitions and Explanations of Terms Unless otherwise defined herein, scientific and technical terms associated with this application have the meanings that are understood by those of ordinary skill in the art.

[0024] Also, unless specifically stated otherwise herein, singular terms herein shall include the plural and plural terms shall include the singular. More specifically, the singular forms "one," "an," and "such," as used in this specification and the appended claims, include plural referents unless clearly stated otherwise.

[0025] The terms "comprise", "include", and "have" are used interchangeably herein and are intended to indicate the inclusiveness of a scheme, meaning that there may be other elements in the scheme than the recited elements. At the same time, it should be understood that the statements "comprise", "include", and "have" as used herein also provide a scheme "consisting of".

[0026] The term "and / or" as used herein includes the meaning of "and", "or" and "all or any other combination of the elements linked by the term to which it pertains".

[0027] In this specification, the term "BCMA" is an abbreviation of B cell maturation antigen, which belongs to a member of the tumor death factor receptor family. BCMA is mainly expressed on the surface of late B cells, short-lived proliferating plasmablasts and long-lived plasma cells, but not on early B cells, CD34-positive hematopoietic stem cells and other normal tissue cells, but is highly expressed in MM cells and plays an important role in the survival, proliferation, metastasis and drug resistance of MM cells by mediating downstream signaling pathways, making BCMA an ideal antigen target for MM treatment.

[0028] As used herein, the term "specific binding" refers to an antigen-binding molecule (e.g., an antibody) that typically specifically binds to an antigen and a substantially identical antigen with high affinity, but does not bind to unrelated antigens with high affinity. Affinity is typically reflected by the equilibrium dissociation constant (KD), where a low KD represents a high affinity. For an antibody as an example, high affinity is typically about 10 -6 M or less, 10 -7 M or less, about 10 -8 M or less, about 10 -9 It means that the KD is equal to or less than M. The calculation formula for KD is as follows: KD=Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis assay.

[0029] As used herein, the term "antigen-binding molecule" is used in the broadest sense and refers to a molecule that specifically binds to an antigen. Exemplary antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refers to organic compounds or binding domains that can specifically bind to an antigen but are unrelated to antibody structure, and exemplary antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0030] As used herein, the term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region and is thereby capable of specifically binding to an antigen. As used herein, "antibody" encompasses a variety of forms and a variety of structures, so long as it exhibits the desired antigen-binding activity. As used herein, "antibody" includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which, for example, contain mutations introduced to stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds, including, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may further include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used for CDR grafting, including, but not limited to, tenascin, fibronectin, peptide aptamers, and the like.

[0031] The term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together via disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions may be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The immunoglobulin heavy chain constant regions differ in amino acid composition and sequence, and therefore in their antigenicity. This allows the "immunoglobulins" herein to be divided into five classes, or may be referred to as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. The same class Ig can be divided into various subclasses depending on the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into κ chains and λ chains depending on the constant region. Each of the five classes of Ig can have either κ chains or λ chains.

[0032] As used herein, "antibody" further includes antibodies that do not contain light chains, for example heavy-chain antibodies (HCAbs) produced by camelids such as Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca (Vicugna pacos), and Ig new antigen receptors (IgNARs) found in chondrichthyes such as sharks.

[0033] As used herein, the term "antibody" may be derived from any animal, including, but not limited to, humans and non-human animals, said non-human animals being selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or chondrichthyes (e.g., sharks).

[0034] As used herein, the term "heavy chain antibody" refers to an antibody lacking the light chain of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain in the absence of a CH1 domain, and CH2 and CH3 constant domains.

[0035] As used herein, the term "nanobody" refers to a single domain antibody consisting only of a heavy chain variable region, obtained by cloning the variable region of a heavy chain antibody (heavy chain antibodies present in animals such as camels, which naturally lack light chains), and is also called VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0036] As used herein, the terms "VHH domain", "nanobody" and "single domain antibody (sdAb)" have the same meaning and are used interchangeably, and refer to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the minimum antigen-binding fragment with complete functionality. Typically, a heavy chain antibody that is naturally missing the light chain and heavy chain constant region 1 (CH1) is obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0037] For further explanation of "heavy chain antibodies", "single domain antibodies", "VHH domains" and "nanobiodies" see: Hamers-Casterman et al., Nature. 1993, 363; 446-8, the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), and the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103, WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193, WO97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527, WO 03 / 050531, WO 01 / 90190, WO03 / 025020, WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787, WO No. 06 / 122825 and other prior art cited in these applications.

[0038] As used herein, the term "multispecific" refers to the ability of an antibody or antigen-binding fragment thereof to bind at least two different epitopes, for example, on different antigens or on the same antigen. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody can bind. For example, a typical monospecific IgG-type antibody has two identical antigen-binding sites (complementary sites), and therefore can only bind to the same epitope (rather than binding to different epitopes). In contrast, a multispecific antibody has at least two different types of complementary sites / binding sites, and therefore can bind to at least two different epitopes. As explained herein, "complementarity determining region" refers to the antigen-binding site of an antibody. Also, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions in a single antibody (the actual number of complementary determining regions / binding sites in one single antibody molecule is referred to as "valency"). For example, a single natural IgG antibody is monospecific and bivalent, since it has two identical complementary sites. Accordingly, a multispecific antibody comprises at least two (different) complementarity determining regions / binding sites. The term "multispecific antibody" therefore refers to an antibody having more than one complementary site and thus the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but also generally includes proteins, e.g. antibodies, scaffolds, i.e. antibodies having three or more complementary sites / binding sites.

[0039] As used herein, the term "valency" refers to the presence of a given number of binding sites on an antibody / antigen-binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites, and six binding sites, respectively, on an antibody / antigen-binding molecule.

[0040] As used herein, "full length antibody," "intact antibody," and "whole antibody" are used interchangeably and refer to having a structure substantially similar to that of a native antibody.

[0041] As used herein, the terms "antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of a whole antibody, but only include local or regional variants of a whole antibody, which have the ability to bind to an antigen. Exemplarily, as used herein, "antigen-binding fragment" or "antibody fragment" includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody, and single domain antibody.

[0042] As used herein, the term "chimeric antibody" refers to an antibody that has immunoglobulin variable sequences derived from one source organism (e.g., rat, mouse, rabbit, or alpaca) and immunoglobulin constant regions derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202, all of which are incorporated herein by reference.

[0043] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to improve homology with the sequence of a human antibody. Typically, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (acceptor antibody). A humanized antibody typically retains or partially retains the desired properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to improve immune cell activity or enhance immune responses, etc.

[0044] As used herein, a "fully human antibody" refers to an antibody having variable regions in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. As used herein, a fully human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific induction or in vivo somatic mutation). However, as used herein, a "fully human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0045] As used herein, "variable region" refers to the region of an antibody's heavy or light chain that is involved in binding the antibody to an antigen, with "heavy chain variable region" being used synonymously with "VH" and "HCVR" and "light chain variable region" being used synonymously with "VL" and "LCVR". The heavy and light chain variable domains of a natural antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0046] As used herein, "complementarity determining region" is used synonymously with "CDR" and generally refers to the hypervariable regions (HVRs) found in both light and heavy chain variable domains. The more conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions that represent the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art. Some positions within a variable domain may be considered to be within the hypervariable region under one set of standards (e.g., IMGT or KABAT), while being outside the hypervariable region under a different set of standards (e.g., KABAT or IMGT), and thus may be considered heterozygous hypervariable positions. One or more of these positions may also be found in an extended hypervariable region. The present invention includes antibodies that include modifications at these heterozygous hypervariable positions. The heavy chain variable region CDRs may be abbreviated as HCDRs and the light chain variable region may be abbreviated as LCDRs. Each of the variable domains of naturally occurring heavy and light chains contains four framework regions that mainly adopt a sheet structure, which are connected through three CDRs (CDR1, CDR2 and CDR3) that form loops connecting, and in some cases forming part of, the sheet structure. The CDRs in each chain are closely held together through the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen-binding site of antibodies with the CDRs from other antibody chains (see Kabat et al., Sequences of Protein sofImmunological Interest, National Institute of Health, Bethesda, Md. 1987, which is incorporated herein by reference).

[0047] Further descriptions of CDRs can be found in Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol., 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc See MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001). As used herein, "CDRs" may be labeled and defined in any manner known in the art, including, but not limited to, the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and tool sites that may be used include, but are not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). As used herein, CDRs include overlapping and subsets of amino acid residues in different defined ways.

[0048] As used herein, the term "Kabat numbering system" generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0049] As used herein, the term "Chothia numbering system" generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical convention for identifying boundaries of CDR regions based on the location of loop regions in the structure (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0050] As used herein, the term "IMGT numbering system" generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) proposed by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0051] As used herein, "heavy chain constant region" refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding an antibody to an antigen but exhibits effector functions such as interaction with an Fc receptor, and has an amino acid sequence more conservative than that of the variable domain of an antibody. The "heavy chain constant region" is selected from a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment", the former having a structure substantially similar to that of the constant region of a natural antibody, while the latter includes only a "part of the full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain, and if the antibody is an IgE, it further includes a CH4 domain, and if the antibody is a heavy chain antibody, it does not include a CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" is selected from an Fc or CH3 domain.

[0052] As used herein, the term "light chain constant region" refers to the carboxyl-terminal portion of an antibody light chain, which is not directly involved in binding the antibody to an antigen, said light chain constant region being selected from a constant κ domain or a constant λ domain.

[0053] As used herein, the term "Fc region" is intended to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Exemplarily, a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by a host cell can undergo post-translational cleavage to remove one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, an antibody produced by a host cell may comprise a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447), may or may not be present. Typically, an IgG Fc region comprises an IgG CH2 and an IgG CH3 domain, and may optionally further comprise a complete or partial hinge region, but does not comprise a CH1 domain. The "CH2 domain" of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises residues at the C-terminus of the CH2 domain in the Fc region (i.e., from an amino acid residue at about position 341 to an amino acid residue at about position 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced in one chain and a corresponding "cavity" or "hole" introduced in another chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference).As described herein, such mutant CH3 domains may be used to promote heterodimerization of two different antibody heavy chains.

[0054] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is based on Kabat et al., Sequences of Proteins of Immunological Interest, 5 th This is based on the EU numbering system, also called the EU index, as described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0055] As used herein, the term "Fc variant" refers to the presence of one or more amino acid substitutions, insertions or deletions at appropriate sites in Fc, resulting in changes in Fc structure or function. "Interaction between Fc variants" refers to the space-filling effect, electrostatic induction, hydrogen bonding effect, and hydrophobic interactions that may be formed between Fc variants designed by mutation. The interaction between Fc variants helps to form a stable heterodimeric protein. The preferred mutation is a "knob-into-hole" type of mutation design.

[0056] Mutational design techniques for Fc variants have been widely applied in the art to generate bispecific antibodies or heterodimeric Fc fusion protein forms. Representative examples include the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp .617-621, 1996); the Fc-containing heterodimer form formed by electrostatic steering by Amgen engineers (US 20100286374 A1); the heterodimer form formed by IgG / Ig chain exchange (SEEDbodies) proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.1-8, 2010); the bispecific molecule formed by Genmab's DuoBody (Science, 2007.317(5844)) platform technology; and the heterodimer protein form formed by Xencor engineers by integrating structural calculations and Fc amino acid mutations to integrate different action modes (mAbs 3:6,546-557; November / December 2011), the heterodimeric protein form obtained by the charge network-based Fc remodeling method (CN201110459100.7) by Suzhou Kangning Jierui Co., Ltd., and other genetic engineering methods to achieve the formation of heterodimeric functional proteins based on Fc amino acid changes or function remodeling means. The knob / hole structure in the Fc variant fragment described in the present invention refers to the fact that the two Fc fragments are mutated respectively and can be bound in the form of "knob-into-hole" after mutation. It is preferable to carry out site mutation remodeling in the Fc region using the "knob-into-hole" model of Cater et al., so that the obtained first Fc variant and second Fc variant can be bound together in the form of "knob-into-hole" to form a heterodimer. It is within the knowledge of a person skilled in the art to select a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass. Preferably, it is the Fc region of human antibody IgG1, IgG2, IgG3, or IgG4, and more preferably, it is the Fc region of human antibody IgG1.A first Fc variant or a second Fc variant is randomly selected, one of which is mutated with a knob and the other with a hole.

[0057] As used herein, the term "conservative amino acids" generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation and rigidity). Illustratively, the following groups of amino acids belong to the conserved amino acid residues of each other, and substitutions of amino acid residues within the groups belong to the conservative amino acid substitutions: 1) Alanine (A), Serine (S), Threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) arginine (R), lysine (K), histidine (H); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0058] As used herein, "identity" is calculated according to the following method: To determine the percentage of "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. Taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap, the percentage of identity between the two sequences varies according to the change in the same position common to the sequences.

[0059] The sequence comparison and identity percentage calculation between two sequences can be realized by using a mathematical algorithm. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm incorporated in the GAP program of the GCG software package (available at www.gcg.com) is used to determine the identity percentage between two amino acid sequences, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. Furthermore, for example, the GAP program of the GCG software package (available at www.gcg.com) is used to determine the identity percentage between two nucleotide sequences, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and that should be used unless otherwise specified) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Additionally, the percent identity between two amino acid or nucleotide sequences can be determined utilizing the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) as implemented in the ALIGN program (version 2.0), using a PAM120 weighting covariance table, a gap length penalty of 12, and a gap penalty of 4.

[0060] Additionally or alternatively, the nucleic acid and protein sequences described in the present invention may be further used as "query sequences" to perform searches against common databases, for example, to identify other family member sequences or related sequences. For example, such searches can be performed with the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches may be performed with the NBLAST program, score=100, wordlength=12, to obtain the same nucleotide sequences as the nucleic acid molecules of the present invention. BLAST protein searches may be performed with the XBLAST program, score=50, wordlength=3, to obtain the same amino acid sequences as the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST may be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0061] As used herein, the term "nucleic acid" includes any compound and / or substance that is a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or riboso) and a phosphate group. A nucleic acid molecule is usually described by the sequence of bases, whereby said bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is usually represented 5' to 3'. As used herein, the term nucleic acid molecule includes, for example, deoxyribose nucleic acid (DNA), including complementary DNA (cDNA) and genomic DNA, ribosomal nucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules may be linear or circular. The term nucleic acid molecule also includes both sense and antisense strands, and single-stranded and double-stranded forms. And the nucleic acid molecules described herein may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules further encompass DNA and RNA molecules suitable for expressing the antibodies of the invention as vectors in vitro and / or in vivo, e.g., directly in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, whereby the mRNA can be injected into a subject to produce the antibody in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP2101823B1).

[0062] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell, which normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0063] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of nucleic acids that are operably linked to them. Such vectors are referred to herein as "expression vectors."

[0064] As used herein, "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and the progeny derived therefrom, without regard to the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid material and may contain mutations. As used herein, mutant progeny that have the same function or biological activity as screened or selected from the initially transformed cell are included.

[0065] As used herein, a "drug composition" refers to a formulation that is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain other ingredients that are unacceptably toxic to a subject to which the drug composition is administered.

[0066] As used herein, "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial and antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drug stabilizers, adhesives, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, which are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pages 1289-1329). Except insofar as it is incompatible with the active ingredient, the use of any conventional carrier in the therapeutic or pharmaceutical compositions is contemplated.

[0067] As used herein, "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions, such as cancer and tumors, in the subject being treated. Beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete remission), whether detectable or undetectable. Subjects in need of treatment include those suffering from a disorder or disease, and those susceptible to a disorder or disease, or those in whom a disorder or disease is to be prevented. When terms such as slow down, alleviation, reduction, mitigation, remission are mentioned, the meaning also includes removal, disappearance, non-occurrence, and the like.

[0068] As used herein, the term "subject" refers to a living organism receiving treatment for a particular disease or disorder as described herein. Exemplary, a "subject" includes a mammal, such as a human, a primate (e.g., a monkey), or a primate mammal, receiving treatment for a disease or disorder.

[0069] As used herein, the term "effective amount" refers to a therapeutic dose that can effectively prevent or ameliorate disease disorders or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. "Effective amount" also refers to a dose of a compound that is sufficient to ameliorate symptoms, for example, treat, cure, prevent, or ameliorate related medical disorders, or increase the rate at which these disorders are treated, cured, prevented, or ameliorated. When an active ingredient is administered alone to an individual, the therapeutically effective dose refers to the ingredient alone. When a combination is applied, the therapeutically effective dose refers to the combined dose of active ingredients that leads to a therapeutic effect, whether combined, administered sequentially, or administered simultaneously.

[0070] As used herein, the term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers. As used herein, "tumor" or "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

[0071] As used herein, the term "EC50" refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between the baseline and maximum after a particular exposure time. EC50 essentially represents 50% of the antibody concentration at which its maximum effect is observed, and can be measured by methods known in the art. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] The present application provides antibodies or antigen-binding fragments thereof that specifically bind to BCMA, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors, host cells, pharmaceutical compositions, kits, methods of manufacture and their applications in the treatment of disease and detection of BCMA.

[0073] In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BCMA, the antibody or antigen-binding fragment thereof comprising CDR1, CDR2 and CDR3, wherein the CDR1 comprises an HCDR1 of a VHH domain shown in any one of SEQ ID NOs: 7 to 21, 24 to 31 and 92 to 101, the CDR2 comprises an HCDR2 of a VHH domain shown in any one of SEQ ID NOs: 7 to 21, 24 to 31 and 92 to 101, and the CDR3 comprises an HCDR3 of a VHH domain shown in any one of SEQ ID NOs: 7 to 21, 24 to 31 and 92 to 101.

[0074] In some specific embodiments, said HCDR1, HCDR2 and HCDR3 are defined according to the Kabat, IMGT or Chothia numbering systems.

[0075] In an optional embodiment, the HCDR1, HCDR2 and HCDR3 are selected from the amino acid sequences set forth in SEQ ID NOs: 34-91 and 102-157.

[0076] In optional embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:34, 37, 40, 42, 45, 48, 50, 52, 54, 56, 58, 64, 67, 71, 74, 77, 81, 84, 86, 87, 104, 107, 113, 116, 119, 121, 124, 127, 131, 134, 138, or 141.

[0077] In optional embodiments, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, 38, 41, 43, 46, 49, 59, 61, 63, 65, 68, 70, 72, 75, 78, 79, 82, 85, 88, 89, 90, 91, 102, 105, 108, 109, 111, 114, 117, 120, 122, 125, 128, 129, 132, 135, 136, 139, 142, 143, 148, 150, 152, 153, 155 or 157.

[0078] In optional embodiments, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, 39, 44, 47, 51, 53, 55, 57, 60, 62, 66, 69, 73, 76, 80, 83, 103, 106, 110, 112, 115, 118, 123, 126, 130, 133, 137, 140, 144, 145, 146, 147, 149, 151, 154 or 156.

[0079] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs:34, 35 and 36, SEQ ID NOs:37, 38 and 39, or SEQ ID NOs:40, 41 and 36, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0080] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 42, 43 and 44, SEQ ID NOs: 45, 46 and 47, or SEQ ID NOs: 48, 49 and 44, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0081] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs:50, 35 and 51, SEQ ID NOs:52, 38 and 53, or SEQ ID NOs:54, 41 and 51, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0082] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:34, 35 and 55, SEQ ID NOs:56, 38 and 57, or SEQ ID NOs:58, 41 and 55, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0083] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:34, 59 and 60, SEQ ID NOs:56, 61 and 62, or SEQ ID NOs:58, 63 and 60, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0084] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:64, 65 and 66, SEQ ID NOs:67, 68 and 69, or SEQ ID NOs:58, 70 and 66, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0085] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs:71, 72 and 73, SEQ ID NOs:74, 75 and 76, or SEQ ID NOs:77, 78 and 73, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0086] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:34, 79 and 80, SEQ ID NOs:81, 82 and 83, or SEQ ID NOs:84, 85 and 80, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0087] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 86, 35 and 36, respectively, according to the Kabat numbering system.

[0088] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 87, 88 and 44, respectively, according to the Kabat numbering system.

[0089] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 42, 88 and 44, respectively, according to the Kabat numbering system.

[0090] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 42, 89 and 44, respectively, according to the Kabat numbering system.

[0091] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 42, 90 and 44, respectively, according to the Kabat numbering system.

[0092] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 42, 91 and 44, respectively, according to the Kabat numbering system.

[0093] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs:71, 102 and 103, SEQ ID NOs:104, 105 and 106, or SEQ ID NOs:107, 108 and 103, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0094] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:71, 109 and 110, SEQ ID NOs:104, 111 and 112, or SEQ ID NOs:107, 108 and 110, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0095] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:113, 114 and 115, SEQ ID NOs:116, 117 and 118, or SEQ ID NOs:119, 120 and 115, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0096] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:121, 122 and 123, SEQ ID NOs:124, 125 and 126, or SEQ ID NOs:127, 128 and 123, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0097] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:113, 129 and 130, SEQ ID NOs:131, 132 and 133, or SEQ ID NOs:134, 135 and 130, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0098] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:113, 136 and 137, SEQ ID NOs:138, 139 and 140, or SEQ ID NOs:141, 142 and 137, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0099] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs:34, 143 and 144, SEQ ID NOs:37, 38 and 145, or SEQ ID NOs:40, 41 and 144, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0100] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences as set forth in SEQ ID NOs:34, 143 and 146, SEQ ID NOs:56, 38 and 147, or SEQ ID NOs:58, 41 and 146, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0101] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs:34, 148 and 149, SEQ ID NOs:37, 150 and 151, or SEQ ID NOs:40, 152 and 149, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0102] In a preferred embodiment, said HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs:71, 153 and 154, SEQ ID NOs:104, 155 and 156, or SEQ ID NOs:107, 157 and 154, respectively, according to the Kabat, IMGT or Chothia numbering systems.

[0103] In some specific embodiments, the CDR1, CDR2 and / or CDR3 comprise an amino acid sequence in which one, two or three mutations occur in said HCDR1, HCDR2 and / or HCDR3, said mutations being selected from insertions, deletions and / or substitutions, said substitutions being preferably conservative amino acid substitutions.

[0104] In some specific embodiments, the CDR1, CDR2 and / or CDR3 comprise an amino acid sequence having at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the HCDR1, HCDR2 and / or HCDR3.

[0105] In some specific embodiments, the antibody or antigen-binding fragment thereof comprises a single domain antibody comprising the aforementioned CDR1, CDR2 and CDR3.

[0106] In some specific embodiments, the single domain antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7-21, 24-31, and 92-101.

[0107] In optional embodiments, the single domain antibody comprises an amino acid sequence in which at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation occurs compared to the sequence set forth in any one of SEQ ID NOs: 7-21, 24-31 and 92-101, wherein the mutations are selected from insertions, deletions and / or substitutions, and the substitutions are preferably conservative amino acid substitutions.

[0108] In optional embodiments, the single domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence set forth in any one of SEQ ID NOs:7-21, 24-31 and 92-101.

[0109] In some specific embodiments, the antibody comprises a FR region in a VHH domain shown in any one of SEQ ID NOs: 7-21, 24-31, and 92-101.

[0110] In an optional embodiment, the antibody comprises an amino acid sequence in which at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation occurs compared to the FR region in the VHH domain shown in any one of SEQ ID NOs: 7 to 21, 24 to 31, and 92 to 101, the mutation being selected from an insertion, deletion, and / or substitution, and the substitution is preferably a conservative amino acid substitution;

[0111] In an optional embodiment, the antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the FR region of the VHH domain shown in any one of SEQ ID NOs:7 to 21, 24 to 31 and 92 to 101.

[0112] In some specific embodiments, the antibody or antigen-binding fragment thereof is selected from: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.

[0113] In some specific embodiments, the antibody or antigen-binding fragment thereof may or may not comprise an antibody heavy chain constant region, optionally the antibody heavy chain constant region is selected from human, alpaca, mouse, rat, rabbit, and sheep, optionally the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE, and IgD, optionally the IgG is selected from IgG1, IgG2, IgG3, and IgG4, optionally the heavy chain constant region is selected from an Fc region, a CH3 region, and a complete heavy chain constant region, preferably the heavy chain constant region is a human Fc region, and preferably the antibody or antigen-binding fragment thereof is a heavy chain antibody.

[0114] In some specific embodiments, the antibody or antigen-binding fragment thereof is further coupled to a therapeutic agent or tracer, preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

[0115] In some specific embodiments, the antibody or antigen-binding fragment thereof is further linked to another functional molecule, preferably selected from one or more of a signal peptide, a protein tag, a cytokine, an angiogenesis inhibitor, and an immune checkpoint inhibitor.

[0116] In a second aspect, the present application further provides a multispecific antigen-binding molecule, said multispecific antigen-binding molecule comprising the aforementioned antibody or antigen-binding fragment thereof, and an antigen-binding molecule that binds to an antigen other than BCMA, or that binds to a different BCMA epitope than the aforementioned antibody or antigen-binding fragment thereof, optionally wherein the antigen other than BCMA is selected from CD3 (preferably CD3ε), CD16, CD137, CD258, PD-1, PD-L1, 4-1BB, CD40, CD64, EGFR, VEGF, HER2, HER1, HER3, IGF-1R, Phosphatidylserine (PS), C-Met, HSA, GPRC5D, MSLN, blood-brain barrier receptor, GPC3, PSMA, CD33, GD2, ROR1, ROR2, FRα and Gucy2C.

[0117] In a preferred embodiment, the antigen-binding molecule is an antibody or an antigen-binding fragment thereof.

[0118] In a preferred embodiment, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific.

[0119] In preferred embodiments, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

[0120] In a third aspect, the present application further provides an isolated nucleic acid fragment, said nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment thereof, or multispecific antigen-binding molecule.

[0121] In a fourth aspect, the present application further provides a vector, said vector comprising the nucleic acid fragment as defined above.

[0122] In a fifth aspect, the present application further provides a host cell, said host cell comprising the above-mentioned vector, preferably said cell is a prokaryotic or eukaryotic cell, such as a bacterial (e.g. E. coli), a fungal (e.g. yeast), an insect cell or a mammalian cell (e.g. a CHO cell line or a 293T cell line).

[0123] In a sixth aspect, the present application further provides a method of producing the above-mentioned antibody or antigen-binding fragment thereof, or multispecific antigen-binding molecule, the method comprising culturing the above-mentioned cell and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell.

[0124] In a seventh aspect, the present application further provides a pharmaceutical composition, the pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, nucleic acid fragment, vector, or product produced according to the above-mentioned method, optionally, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, diluent or adjuvant, and optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0125] In some embodiments, the pharma- ceutically acceptable carrier is a carrier that does not reduce the viability and function of immune cells and does not affect the specific binding between an antibody or an antigen-binding fragment thereof and an antigen, and includes, but is not limited to, cell culture medium, buffer, saline, balanced salt solution, etc. Examples of buffers include isotonic phosphate, acetate, citrate, borate, carbonate, etc. In a specific embodiment, the pharma-ceutically acceptable carrier is a phosphate buffer containing 1% serum.

[0126] The antitumor agent may be, for example, various chemotherapeutic agents, such as alkylating agent chemotherapeutic agents, including but not limited to, cyclophosphamide, isocyclophosphamide, melphalan, carmustine, lomustine, nimustine, fotemustine, and the like; antimetabolite chemotherapeutic agents, including but not limited to, methotrexate, fluorouracil, tegafur, carmofur, fluorodeoxyuridine, capecitabine, gemcitabine, raltitrexed, and the like; actinomycin A, B, C, D, E, F, G, H, I, M, M, N, O, P, R, T ... Anticancer antibiotics, including but not limited to, syn D, mitomycin, bleomycin, daunorubicin, doxorubicin, epirubicin, pyrandoxorubicin, etc.; plant-based chemotherapeutic drugs, including but not limited to, vincristine, vindesine, vinorelbine, irinotecan, topotecan, paclitaxel, docetaxel, etc.; miscellaneous drugs, including but not limited to, dacarbazine, cisplatin, carboplatin, oxaliplatin, nedaplatin, etc.

[0127] In an eighth aspect, the present application further provides a method of treating a tumor or cancer, the method comprising administering to a subject an effective amount of said antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, nucleic acid fragment, vector, product manufactured according to said method, or said pharmaceutical composition, optionally wherein said tumor or cancer is a BCMA-expressing tumor or cancer, preferably a B-cell lymphoma, more preferably multiple myeloma (MM).

[0128] In a ninth aspect, the present application further provides the use of said antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, nucleic acid fragment, vector, product manufactured according to said method or pharmaceutical composition in the manufacture of a medicament for treating a tumor or said cancer, optionally wherein said tumor or cancer is a BCMA-expressing tumor or cancer, preferably a B-cell lymphoma, more preferably multiple myeloma (MM).

[0129] In a tenth aspect, the present application further provides a use of said antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, nucleic acid fragment, vector, product manufactured according to said method or said pharmaceutical composition for treating a tumor or cancer, optionally wherein said tumor or cancer is a BCMA-expressing tumor or cancer, preferably a B-cell lymphoma, more preferably multiple myeloma (MM).

[0130] In an eleventh aspect, the present application further provides a kit, said kit comprising the above-mentioned antibody or antigen-binding fragment thereof, multispecific antigen-binding molecule, nucleic acid fragment, vector, product produced according to the above-mentioned method, or the above-mentioned pharmaceutical composition.

[0131] In a twelfth aspect, the present application further provides a method of detecting BCMA expression in a biological sample, said method comprising contacting said biological sample with said antibody or antigen binding fragment thereof under conditions allowing the formation of a complex between said antibody or antigen binding fragment thereof and BCMA, preferably said method further comprising detecting the formation of said complex, indicating the presence or expression level of BCMA in the sample.

[0132] In a thirteenth aspect, the present application further provides the use of the aforementioned antibody or antigen-binding fragment thereof in the manufacture of a BCMA detection reagent.

[0133] Working Example In the following, the present application will be further described with reference to specific examples, and the advantages and features of the present application will be apparent from the description. If no specific conditions are specified in the examples, they will be carried out according to conventional conditions or conditions suggested by the manufacturer. If no manufacturer is specified, the reagents or equipment used are all general products that can be purchased commercially.

[0134] The examples in this specification are merely illustrative and do not limit the scope of the present application. Those skilled in the art will understand that modifications or replacements can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and all of these modifications and replacements are within the protection scope of the present application.

[0135] Example 1 Production of control antibodies, identification of endogenous cells and production of overexpressing cell lines (A) Preparation of control antibody REGN5459 and HPN217 are both antibodies that recognize human BCMA protein, where the REGN5459 sequence is derived from US Patent Publication No. US2020 / 0024356A1, and the HPN217 sequence is derived from US Patent Publication No. US20190112381A1. The heavy chain variable region (VH) of REGN5459 was recombined into an expression vector containing a signal peptide and a human-derived antibody IgG1 heavy chain constant region, and the light chain variable region (VL) sequence was recombined into an expression vector containing a signal peptide and a human-derived antibody IgG1 light chain constant region to obtain a recombinant plasmid, and the synthesized antibody was named REGN5459-hIgG1. The VHH sequence of HPN217 was recombined into an expression vector containing a signal peptide and a human-derived antibody IgG1 Fc to obtain a recombinant plasmid, and the synthesized antibody was named HPN217-hHcAb. The negative control antibody hIgG1 was an antibody against Hen Egg Lysozyme, anti-hel-hIgG1 (purchased from Hyakuei, catalog number: B117901), hereinafter abbreviated as hIgG1.

[0136] [Table 1]

[0137] The binding activity of the control antibody to human BCMA-His protein (purchased from Acro, catalog number: BCA-H522y) and monkey BCMA-His protein (purchased from Acro, catalog number: BCA-C52H7) was detected by ELISA. The specific method is as follows: the antigen protein was diluted with PBS to a final concentration of 1 μg / mL, and added to a 96-well ELISA plate at 50 μl / well. It was sealed with plastic film and incubated overnight at 4°C. The next day, the plate was washed twice with PBS, and a mounting solution [PBS + 2% (w / v) BSA] was added and blocked at room temperature for 2 hours. The mounting solution was discarded, and 100 nM of gradient-diluted control antibody or negative control antibody was added at 50 μl / well. After incubation at 37°C for 2 hours, the plate was washed three times with PBS. Secondary antibody labeled with HRP (horseradish peroxidase) (purchased from Merck, catalog number: AP113P) was added and incubated at 37°C for 1 hour, after which the plate was washed 5 times with PBS. TMB substrate was added at 50μl / well and incubated at room temperature for 10 minutes, after which stop solution (1.0M HCl) was added at 50μl / well. OD450nm was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The results are shown in Tables 2 and 3 and Figures 1A and 1B, and the REGN5459-hIgG1 antibody has good binding activity to both human BCMA protein and monkey BCMA protein, and HPN217-hHcAb has good binding activity to human BCMA protein but does not bind to monkey BCMA protein.

[0138] [Table 2]

[0139] [Table 3]

[0140] (B) Identification of cell lines that endogenously express BCMA protein. H929 and U266 cells were cultured in a T-75 cell culture flask until logarithmic growth phase, and the cells were pipetted to form a single cell suspension. After counting the cells, they were centrifuged, and the cell pellet was diluted to 2 × 10 with FACS buffer (PBS + 2% fetal bovine serum). 6 The cells were resuspended to cells / milliliter and added to a 96-well FACS reaction plate at 50 μl / well, and detected and analyzed by FACS (FACS Canto™, purchased from BD) using REGN5459-hIgG1 and HPN217-hHcAb antibodies as primary antibodies and goat anti-human IgG (H+L) labeled with APC as secondary antibody (purchased from Jackson, catalog number: 109-605-088). The results are shown in Table 4 and Figures 2A and 2B, and show that H929 cells and U266 cells can bind to both REGN5459-hIgG1 and HPN217-hHcAb antibodies.

[0141] [Table 4]

[0142] (C) Production of Flp-inCHO recombinant cell lines of human and monkey BCMA proteins The nucleotide sequences encoding the human BCMA and monkey BCMA amino acid sequences were cloned into the pcDNA5 / FRT vector (purchased from Clontech), respectively: human BCMA NCBI Gene ID: 608, monkey BCMA NCBI Gene ID: 712212. Flp-inCHO cell line (purchased from the Cell Bank of the Committee for the Depository of Typical Cultures, Chinese Academy of Sciences) was transfected (Lipofectamine® 3000 Transfection Kit, purchased from Invitrogen, Catalog No. L3000-015) and selectively cultured in F12K Medium (Gibco, Catalog No. 21127030) containing 10% (v / v) fetal bovine serum (ExCell Bio, Catalog No. FND500) containing 600 μg / ml hygromycin (ThermoFisher, Catalog No. 10687010) for 2 weeks, and analyzed by flow cytometer FACS CantoII (BD) using REGN5459-hIgG1 and goat anti-human IgG (H+L) antibodies (Jackson, Catalog No. 109605088). The expression level was detected by ELISA using a 200-ml IgG antibody (purchased from Biosciences), and cells with high expression and single peak shape were amplified, and the amplified cells were re-detected by flow cytometry analysis. A positive cell population with good growth, high fluorescence intensity, and high uniformity was selected and expanded, and then cryopreserved in liquid nitrogen. Table 5 shows that a human BCMA-positive expression Flp-inCHO high expression cell population and a monkey BCMA-positive expression Flp-inCHO high expression cell population were produced, respectively. In Figures 3 and 4, the abscissa is cell fluorescence intensity, and the ordinate is cell number.

[0143] [Table 5]

[0144] Example 2: Production of alpaca nanobody against BCMA (A) Animal immunization and serum titer measurement The immunized animal was one alpaca (Alpaca, No. NB254). 10 mL of blood was collected before immunization to serve as a negative control. For the first immunization, 0.5 mg of human BCMA (Met1-Ala54)-hFc protein (purchased from Acro, catalog number: BC7-H5254) was mixed with 1 mL of Freund's complete adjuvant (CFA) and injected subcutaneously. For the second immunization on the 21st day, 0.25 mg of human BCMA-hFc protein was mixed with 1 mL of Freund's incomplete adjuvant (IFA) and injected subcutaneously. For the third immunization on the 42nd day, 0.25 mg of human BCMA-hFc protein and 0.25 mg of cynomolgus monkey BCMA (Met1-Ala53)-hFc protein (purchased from Acro, catalog number: BCA-C5253) were injected subcutaneously. After homogeneously mixing with 1 mL, the mixture was injected subcutaneously, and then booster immunization was performed every 3 weeks according to the method for the third immunization.

[0145] Blood was collected one week after each booster immunization, and serum antibody titer and specificity were detected by ELISA, and the results are shown in Table 6. The sera of alpacas immunized with human BCMA-hFc and cynomolgus monkey BCMA-hFc proteins were both shown to bind to the immunogen to different degrees, and exhibited antigen-antibody reactions. Here, the blank control group was 1% (w / v) BSA, and the 4th immunization and 5th immunization refer to the alpaca sera on the 7th day after the 4th and 5th immunizations, respectively. The data in the table are OD450nm values, and k represents 1000.

[0146] [Table 6]

[0147] (B) Construction of phage library PBMCs were isolated from 50 mL of peripheral blood collected after the fourth and fifth immunizations according to the instructions for the lymphocyte isolation solution. Total RNA was extracted from PBMCs from the fourth and fifth immunizations of NB254 using RNAiso Plus reagent. PrimeScript TMThe cDNA was produced by reverse transcription using the II 1st Strand cDNA Synthesis Kit (Takara, Catalog No.: 6210A) kit, and reverse transcription was performed according to the instruction manual, and a total of 5 μg of RNA was transcribed. The cDNA stock solutions from the 4th and 5th immunizations were mixed in the same ratio, then diluted 5-fold for nested amplification, and the VHH fragments were recovered by tapping. The vector and the target fragment were each subjected to enzymatic cleavage with SfiI at 50°C overnight, and then recovered. The vector and the target fragment were ligated at a ratio of 1:3. The ligated product was transformed by electroporation 10 times, and immediately after electroporation, 1 mL of 2YT medium (preheated at 37°C) was added to the electroporation cup to resuscitate it, the electroporation product was sucked out, the electroporation cup was washed with 2YT medium, and resuscitated at 37°C and 180 rpm for 45 min, 100 μL was taken, and 10 -3 and 10 -4 The number of transformants in the library was determined by gradient dilution to 100 μl, plated onto a 90 mm plate, and the remainder was centrifuged, resuspended in 8 mL of 2YT medium, and plated onto eight 200 mm plates. The next day, the library volume was calculated to be 2.14 × 10 9 48 clones were randomly selected and cultured, and the insertion rate was detected by PCR and found to be 100%.

[0148] The E. coli library was placed in 2 x 300 mL of 2YT (Amp: 100 μg / ml, Glu: 1%) medium until its initial OD600 = 0.1-0.2, and cultured at 37 °C, 230 rpm until OD600 = 0.8 or more. Based on the OD600, the amount of helper phage M13K07 added (M13K07: E. coli = 20:1) was calculated, and the mixture was left to infect at 37 °C for 30 min, and then cultured at 37 °C, 180 rpm for 30 min. The supernatant was removed by centrifugation at 5000 rpm, and the 2YT (100 μg / mL Amp + 50 μg / mL Kan) medium was replaced and cultured overnight at 30 °C, 220 rpm. The phage library was precipitated according to the procedure on the NEB official website, and its titer was measured.

[0149] (C) Panning against BCMA nanoantibodies Microwell plates were coated with human BCMA-hFc protein at 0.5 μg / well for a total of 8 wells and an input of phage library of 2.2 × 10 11 and the titer of the first round elution product was 1.85 × 10 7 The ELISA identification showed that the first round panning product had a positive binding rate of 89.58% to human BCMA protein, a positive binding rate of 4.2% to monkey BCMA protein, and a positive cross-binding rate of 4.2% to human and monkey BCMA proteins. The first round panning elution product was amplified by a general procedure, and the titer of the amplified phage was 1.14 × 10 13 In the second round of panning, microwell plates were coated with monkey BCMA protein at 0.2 μg / well for a total of 4 wells, and the input of the phage library was 2.2 × 10 11 and the titer of the eluted product was 1.32 × 10 7 As determined by ELISA, the second round panning products showed a 92.1% positive binding rate to human BCMA protein, a 39.3% positive binding rate to monkey BCMA protein, and a 39.3% positive cross-reaction rate to both human and monkey BCMA proteins.

[0150] (D) Sequencing A total of 70 human-monkey cross-positive clones were obtained through panning, and sequencing analysis was performed on the positive clones. A phylogenetic tree sequence was constructed based on the VHH-encoding protein sequences. Clustering analysis revealed that the 70 sequences belonged to three germ lines. A total of eight clones, Lab01, 02, 03, 04, 05, 06, 07 and 08, were selected, constructed, expressed and identified.

[0151] Example 3 Construction and expression of recombinant candidate antibodies The target VHH sequence was recombined into an expression vector for human IgG1 Fc to obtain a recombinant plasmid (see SEQ ID NO: 4 for the sequence of human IgG1 Fc). The recombinant candidate antibody plasmid and transfection reagent PEI (purchased from Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed uniformly, and then left to stand for 15 min., and added to Expi293F cells (manufacturer: Thermofisher, catalog number: A14527), and cultured in a shaker at 5% CO2, 120 rpm, and 37°C. The day after transfection, OPM-293 ProFeed (Shanghai Aopumai, catalog number: F081918-001) and 6 g / L glucose (manufacturer: Sigma, catalog number: G7528) were added. On the 6th day after transfection, the cell supernatant was collected. The supernatant was collected, cultured, and purified by Protein A affinity. The filler used was Mabselect SuRe (No. 10054120, purchased from GE Healthcare), and the buffers were Mab affinity equilibration solution (PBS Buffer), Mab affinity washing solution (PBS buffer containing 1M NaCl), and Mab affinity elution solution (citrate buffer). Concentration was measured using an ultramicrospectrophotometer (Nanodrop8000, Thermo). The protein samples to be measured were analyzed by SEC-HPLC to characterize the molecular size uniformity and purity of the proteins. The HPLC used was Agilent 1260, the chromatography column TSKgel G3000SWXL was purchased from Tosoh Bioscience, the mobile phase was 200 mM phosphate buffer, pH 7.0 / isopropyl alcohol (v / v 9:1) (lot number: 20210519001), the detection temperature was 25°C, the flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. SEC-HPLC data was analyzed by manual integration method to analyze the chromatogram, and the purity of the protein was calculated according to the area normalization method, where the main peak was considered to be the monomer, the chromatographic peak before the main peak was referred to as aggregate, and the chromatographic peak after the main peak was referred to as fragment.The results are shown in Table 7, and the purity of the final candidate antibody product was high.

[0152] [Table 7]

[0153] Example 4 Identification of recombinant candidate antibodies (A) Binding of BCMA recombinant candidate antibodies to human BCMA protein and cross-linking to monkey BCMA protein detected by enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: Human BCMA protein (purchased from Acro, catalog number: BCA-H522y) and monkey BCMA protein (purchased from Acro, catalog number: BCA-C52H7) were each diluted with PBS to a final concentration of 1 μg / mL, and added to a 96-well ELISA plate at 50 μl / well. The plate was sealed with plastic film and incubated overnight at 4°C. The next day, the plate was washed twice with PBS, and a mounting solution [PBS + 2% (w / v) BSA] was added and blocked at room temperature for 2 hours. The mounting solution was discarded, the plate was washed three times with PBS, and 100 nM gradient diluted BCMA nano recombinant chimeric antibody or negative control antibody was added at 50 μl / well. After incubation at 37°C for 2 hours, the plate was washed three times with PBS. Secondary antibody labeled with HRP (horseradish peroxidase) (purchased from Merck, catalog number: AP113P) was added and incubated at 37°C for 1 hour, after which the plate was washed 5 times with PBS. TMB substrate was added at 50 μl / well and incubated at room temperature for 10 minutes, after which stop solution (1.0M HCl) was added at 50 μl / well. OD450nm was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The results are shown in Figures 5 and 6. All antibodies bound to human BCMA protein, BCMA-Lab06 and BCMA-Lab07 had no binding activity to monkey BCMA protein, and the other recombinant candidate antibodies all had cross-binding activity to monkey BCMA protein, and the IgG subtype control was human IgG1.

[0154] (B) Flow cytometry (FACS) detection of binding of BCMA recombinant candidate antibodies to BCMA-expressing endogenous cells. The required cells were expanded to logarithmic growth phase in a T-75 cell culture flask and the cells were pipetted to obtain a single cell suspension. After counting the cells, they were centrifuged and the cell pellet was diluted with FACS buffer (PBS + 2% fetal bovine serum) to 2 × 10 6The cells were resuspended to cells / milliliter and added to a 96-well FACS reaction plate at 50 μl / well, and the candidate antibody test sample was added at 50 μl / well and incubated at 4°C for 1 hour. The cells were centrifuged and washed three times with PBS buffer, and secondary antibody anti-hIgG(Fc)Alexa 647 (purchased from Jackson, catalog number: 109-605-098) was added at 50 μl / well and incubated on ice for 1 hour. The cells were centrifuged and washed three times with PBS buffer, and 100 μl was taken and detected by FACS (FACS CantoTM, purchased from BD), and the results were analyzed. The data was analyzed by software (FlowJo) to obtain the mean fluorescence intensity (MFI) of the cells. The data was then analyzed by software (GraphPad Prism8), data fitting was performed, and EC50 was calculated. The results are shown in Table 8 and Figures 7 and 8. BCMA-Lab05, BCMA-Lab06, and BCMA-Lab08 bound poorly to H929 and U266 cells, while the other antibodies performed well.

[0155] [Table 8]

[0156] (C) Blocking effect of recombinant candidate antibodies on the binding of the ligand APRIL to human BCMA protein detected by competitive ligand binding ELISA. Antigen proteins were diluted with PBS to a final concentration of 1 μg / mL and added to a 96-well ELISA plate at 50 μl / well. Sealed with plastic film and incubated overnight at 4°C, the next day the plate was washed twice with PBS, and mounting solution [PBS + 2% (w / v) BSA] was added and blocked at room temperature for 2 hours. The mounting solution was discarded, the plate was washed three times with PBS, and 200 nM gradient diluted BCMA nano recombinant chimeric antibody or negative control antibody was added at 50 μl / well. hAPRIL-biotin (purchased from Acro, Catalog No.: APL-H82F5) was diluted to 0.5 μg / mL, 50 μl / well. After incubation at 37°C for 1.5 hours, the plate was washed three times with PBS. Secondary antibody labeled with HRP (horseradish peroxidase) (purchased from Sigma, catalog number: S2438-250μg) was added and incubated at 37℃ for 1 hour, then the plate was washed 5 times with PBS. TMB substrate was added at 50μl / well, incubated at room temperature for 10 minutes, and stop solution (1.0M HCl) was added at 50μl / well. OD450nm was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The results are shown in Figure 9, where BCMA-Lab05, BCMA-Lab06 and BCMA-Lab08 have normal blocking effects, while the other antibodies have good blocking effects.

[0157] Example 5. Affinity detection of BCMA recombinant candidate antibodies Anti-BCMA recombinant candidate antibodies were captured using a Protein A chip (GE Healthcare; 29-127-558). Sample and running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69). The flow cell was set at 25°C. The sample block was set at 16°C. Both were pretreated with running buffer. In each cycle, the target antibody was first captured with the Protein A chip, and then a single concentration of BCMA antigen protein was injected, and the binding and dissociation process between the antibody and the antigen protein was recorded, and finally the chip was regenerated with Glycine pH 1.5 (GE Healthcare; BR-1003-54). Binding was measured by injecting different concentrations of human BCMA-His in solution for 240 seconds, with a flow rate of 30 μL / min, starting from 200 nM (see detailed results for actual concentrations tested) and diluted 1:1, for a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds and was triggered by switching from sample solution to running buffer. The surface was regenerated by washing with a 10 mM glycine solution (pH 1.5) at a flow rate of 30 μL / min for 30 seconds. Differences in bulk refractive index were corrected by subtracting the response obtained from the goat anti-human Fc surface. Blank injections (= double references) were also subtracted. A Langmuir 1:1 model was used to calculate the apparent KD and other kinetic parameters. The binding rate (K a ), dissociation rate (K dis ) and binding affinity (KD) are shown in the table, where REGN5459-hIgG1 antibody was used as a control. As shown in Table 9, the KD values ​​of the recombinant candidate antibodies and human BCMA protein are all 1E-7M or less.

[0158] [Table 9]

[0159] After screening through a series of experiments, BCMA-Lab01 and BCMA-Lab02 alpaca antibodies were selected for subsequent humanization. The sequences of BCMA-Lab01 to BCMA-Lab08 alpaca antibodies are shown in Table 10, and the CDR sequences analyzed by Kabat, IMGT, and Chothia methods are shown in Table 11.

[0160] [Table 10]

[0161] [Table 11]

[0162] Example 6 Humanization of Nanobodies By aligning the IMGT (http: / / imgt.cines.fr) germline gene data bank of human antibody heavy and light chain variable regions, the germline genes of heavy chain variable regions with high homology to VHH nanobodies were selected as templates, and the CDRs of VHH nanobodies were grafted onto the corresponding human-derived templates to form the variable region sequences, which are FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in order. If necessary, key amino acids in the framework sequence were back-mutated to form the amino acids corresponding to VHH nanobodies to ensure the original affinity, and a humanized monoclonal antibody was obtained. Here, the CDR amino acid residues of the antibody were determined and annotated according to the Kabat numbering system.

[0163] (A) Humanization of BCMA-Lab01 antibody The humanized heavy chain templates of VHH nanobody BCMA-Lab01 are IGHV3-64*04 and IGHJ3*01, and the CDRs of VHH nanobody BCMA-Lab01 can be grafted onto their respective human-derived templates to obtain the corresponding humanized versions. If necessary, key amino acids in the FR region sequence of the humanized antibody of BCMA-Lab01 can be back-mutated to form the corresponding amino acids of the VHH nanobody to ensure the original affinity; there are sites in the antibody that are prone to chemical modification, and point mutations can be performed on these sites to eliminate the modification risk. The specific mutation design is shown in Table 12.

[0164] [Table 12]

[0165] The specific sequences of the variable regions of the BCMA-Lab01 humanized antibody are as follows: The BCMA-Lab01-VHH1 amino acid sequence is as shown in SEQ ID NO:15: [ka] The BCMA-Lab01-VHH2 amino acid sequence is as shown in SEQ ID NO:16: [ka] The BCMA-Lab01-VHH3 amino acid sequence is as shown in SEQ ID NO:17: [ka] The BCMA-Lab01-VHH4 amino acid sequence is as shown in SEQ ID NO:18: [ka] The BCMA-Lab01-VHH5 amino acid sequence is as shown in SEQ ID NO:19: [ka] The BCMA-Lab01-VHH6 amino acid sequence is as shown in SEQ ID NO:20: [ka] The BCMA-Lab01-VHH7 amino acid sequence is as shown in SEQ ID NO:21: [ka] The humanized heavy chain template IGHV3-64*04 amino acid sequence is as shown in SEQ ID NO:22: [ka] The humanized heavy chain template IGHJ3*01 amino acid sequence is as shown in SEQ ID NO:23: [ka] According to the Kabat numbering system, the analysis results of the above humanized Nanobody VHH sequences are shown in Table 13.

[0166] [Table 13]

[0167] (B) Humanization of BCMA-Lab02 antibody The humanized heavy chain templates of the VHH single domain antibody BCMA-Lab02 are IGHV3-7*01 and IGHJ6*01, and the CDRs of the VHH single domain antibody BCMA-Lab02 are grafted onto the human-derived templates to obtain the corresponding humanized versions. If necessary, key amino acids in the FR region sequence of the humanized antibody of BCMA-Lab02 are back-mutated to form the amino acids corresponding to the VHH single domain antibody to ensure the original affinity, and there are sites in the antibody that are prone to chemical modification, and point mutations are performed on these sites to eliminate the modification risk. The specific mutation design is shown in Table 14.

[0168] [Table 14]

[0169] The specific sequences of the variable regions of the BCMA-Lab02 humanized antibody are as follows: The BCMA-Lab02-VHH1 amino acid sequence is as shown in SEQ ID NO:24: [ka] The BCMA-Lab02-VHH2 amino acid sequence is as shown in SEQ ID NO:25: [ka] The BCMA-Lab02-VHH3 amino acid sequence is as shown in SEQ ID NO:26: [ka] The BCMA-Lab02-VHH4 amino acid sequence is as shown in SEQ ID NO:27: [ka] The BCMA-Lab02-VHH5 amino acid sequence is as shown in SEQ ID NO:28: [ka] The BCMA-Lab02-VHH6 amino acid sequence is as shown in SEQ ID NO:29: [ka] The BCMA-Lab02-VHH7 amino acid sequence is as shown in SEQ ID NO:30: [ka] The BCMA-Lab02-VHH8 amino acid sequence is as shown in SEQ ID NO:31: [ka] The humanized heavy chain template IGHV3-7*01 amino acid sequence is as shown in SEQ ID NO:32: [ka] The humanized heavy chain template IGHJ6*01 amino acid sequence is as shown in SEQ ID NO:33: [ka] According to the Kabat numbering system, the analysis of the above humanized single domain antibody VHH sequences is shown in Table 15.

[0170] [Table 15]

[0171] Example 7 Construction, expression and purity determination of candidate humanized nanobodies For the specific operation method, see Example 3. The target humanized VHH sequence was recombined into the expression vector of human IgG1 Fc to obtain a recombinant plasmid (for the sequence of human IgG1 Fc, see SEQ ID NO:4). The humanized VHH-Fc chimeric antibody was expressed and collected to obtain the cell supernatant. The purity of the obtained antibody was qualitatively analyzed by SEC-HPLC, and the results are shown in Table 16, where " / " indicates not detected.

[0172] [Table 16]

[0173] Example 8 Identification of BCMA humanized candidate nanobodies (A) Binding of BCMA humanized candidate nanobodies to human BCMA protein and cross-linking with monkey BCMA protein detected by enzyme-linked immunosorbent assay (ELISA). The specific method is the same as in Example 4(A), and the results are shown in Figures 10 and 11. BCMA-Lab02-VHH1 and BCMA-Lab02-VHH2 do not bind to either human BCMA or monkey BCMA protein, while the remaining humanized candidate nanobodies all bind well to human BCMA and have cross-binding activity with monkey BCMA protein, and the IgG subtype control is human IgG1.

[0174] (B) Binding of humanized candidate nanobodies to BCMA-expressing endogenous cells detected by flow cytometry (FACS). The specific method was the same as in Example 4(B), and the results are shown in Table 17 and Figures 12 and 13. BCMA-Lab02-VHH1 and BCMA-Lab02-VHH2 did not bind to H929 and U266 cells, while the remaining humanized candidate nanobodies bound well to H929 and U266 cells.

[0175] [Table 17]

[0176] (C) Blocking effect of humanized candidate nanobodies on the binding of the ligand APRIL to human BCMA protein detected by competitive ligand binding ELISA. The specific method is the same as in Example 4(C), and the results are shown in Figure 14, in which BCMA-Lab02-VHH1 and BCMA-Lab02-VHH2 are unable to block the binding between the ligand APRIL and human BCMA protein, BCMA-Lab01VHH1 and BCMA-Lab01VHH2 have poor blocking effect on the binding between the ligand APRIL and human BCMA protein, and the remaining humanized candidate nanobodies have good blocking effect.

[0177] Example 9. Affinity detection of BCMA humanized candidate nanobodies For specific operation methods, please refer to Example 5. The binding rate (Ka), dissociation rate (Kdis) and binding affinity (KD) of the candidate humanized nanobody with human BCMA protein are shown in Table 18, where REGN5459-hIgG1 antibody is used as a control. As shown in Table 18, the KD values ​​of the candidate humanized nanobody with human BCMA protein are all below 1E-8M.

[0178] [Table 18]

[0179] Example 10. Panning of Nanobodies Against BCMA and Identification of Recombinant Candidate Antibodies (A) Panning of nanobodies against BCMA According to the method of Example 2(C), the positive clones that bind to human BCMA were further panned, and a total of 10 clones, Lab09-18, were selected, constructed, expressed, and identified.

[0180] (B) Construction and expression of recombinant candidate antibodies According to the method in Example 3, the recombinant candidate antibodies of VHH-IgG1 were obtained and identified by SEC-HPLC, and the purity of the final candidate antibodies was high.

[0181] (C) Binding of BCMA recombinant candidate antibodies to human BCMA protein detected by ELISA. Referring to the method in Example 4(A), ELISA was used to detect the binding of the recombinant candidate antibodies to human BCMA, and the results are shown in Figure 15. Figure 15 shows that all antibodies bind to human BCMA protein and are comparable to the positive control antibody HPN217-hHcAb.

[0182] (D) Detection of binding of BCMA recombinant candidate antibodies to BCMA-expressing endogenous cells by FACS. Following the method in Example 4(B), FACS was used to identify the binding of the recombinant candidate antibodies to BCMA-expressing endogenous cells, and the results are shown in Figures 16A, 16B and Table 19. The results show that, except for BCMA-Lab09, which binds weakly to H929 and does not bind to U266, the remaining antibodies bind strongly to both H929 and U266 cells, either stronger than or equivalent to the positive control antibody.

[0183] [Table 19]

[0184] (E) Blocking effect of recombinant candidate antibodies on the binding of the ligand APRIL to human BCMA protein detected by competitive ligand binding ELISA. According to the method in Example 4(C), the blocking effect of the recombinant antibodies on the binding of the ligand APRIL to human BCMA protein was detected by competitive ELISA, and the results are shown in Figure 17. The results show that, except for BCMA-Lab09, which cannot block the binding of the ligand APRIL to human BCMA protein, the remaining antibodies can all successfully block the binding of the ligand APRIL to human BCMA protein, which is comparable to the positive control antibody.

[0185] Example 11 Affinity detection of BCMA recombinant candidate antibodies According to the method in Example 5, the affinity of the recombinant antibodies with human BCMA protein was detected by Biacore, and the results are shown in Table 20. The results show that all the antibodies have strong affinity with human BCMA protein. The sequences of the nanobodies are shown in Table 21, and their CDR sequences are shown in Table 22.

[0186] [Table 20]

[0187] [Table 21]

[0188] [Table 22-1] [Table 22-2]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to BCMA, the antibody or antigen-binding fragment thereof comprising CDR1, CDR2 and CDR3, wherein the CDR1 comprises an HCDR1 of a VHH domain shown in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101, the CDR2 comprises an HCDR2 of a VHH domain shown in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101, and the CDR3 comprises an HCDR3 of a VHH domain shown in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101.

2. The HCDR1, HCDR2 and HCDR3 are determined based on the Kabat, IMGT or Chothia numbering system; Optionally, the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 42, 34, 37, 40, 45, 48, 50, 52, 54, 56, 58, 64, 67, 71, 74, 77, 81, 84, 86, 87, 104, 107, 113, 116, 119, 121, 124, 127, 131, 134, 138, or 141; Optionally, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 90, 35, 38, 41, 43, 46, 49, 59, 61, 63, 65, 68, 70, 72, 75, 78, 79, 82, 85, 88, 89, 91, 102, 105, 108, 109, 111, 114, 117, 120, 122, 125, 128, 129, 132, 135, 136, 139, 142, 143, 148, 150, 152, 153, 155, or 157; Optionally, the HCDR3 comprises an amino acid sequence set forth in SEQ ID NO: 44, 36, 39, 47, 51, 53, 55, 57, 60, 62, 66, 69, 73, 76, 80, 83, 103, 106, 110, 112, 115, 118, 123, 126, 130, 133, 137, 140, 144, 145, 146, 147, 149, 151, 154, or 156; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 42, 90 and 44, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 35 and 36, SEQ ID NO: 37, 38 and 39, or SEQ ID NO: 40, 41 and 36, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 42, 43 and 44, SEQ ID NO: 45, 46 and 47, or SEQ ID NO: 48, 49 and 44, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 50, 35 and 51, SEQ ID NOs: 52, 38 and 53, or SEQ ID NOs: 54, 41 and 51, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 35 and 55, SEQ ID NO: 56, 38 and 57, or SEQ ID NO: 58, 41 and 55, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 59 and 60, SEQ ID NO: 56, 61 and 62, or SEQ ID NO: 58, 63 and 60, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 64, 65 and 66, SEQ ID NO: 67, 68 and 69, or SEQ ID NO: 58, 70 and 66, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 71, 72 and 73, SEQ ID NOs: 74, 75 and 76, or SEQ ID NOs: 77, 78 and 73, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 34, 79 and 80, SEQ ID NOs: 81, 82 and 83, or SEQ ID NOs: 84, 85 and 80, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 86, 35 and 36, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 87, 88 and 44, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 42, 88 and 44, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 42, 89 and 44, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 42, 91 and 44, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 71, 102 and 103, SEQ ID NO: 104, 105 and 106, or SEQ ID NO: 107, 108 and 103, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 71, 109 and 110, SEQ ID NO: 104, 111 and 112, or SEQ ID NO: 107, 108 and 110, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 113, 114 and 115, SEQ ID NO: 116, 117 and 118, or SEQ ID NO: 119, 120 and 115, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 121, 122 and 123, SEQ ID NO: 124, 125 and 126, or SEQ ID NO: 127, 128 and 123, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 113, 129 and 130, SEQ ID NOs: 131, 132 and 133, or SEQ ID NOs: 134, 135 and 130, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 113, 136 and 137, SEQ ID NO: 138, 139 and 140, or SEQ ID NO: 141, 142 and 137, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 143 and 144, SEQ ID NO: 37, 38 and 145, or SEQ ID NO: 40, 41 and 144, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 143 and 146, SEQ ID NO: 56, 38 and 147 or SEQ ID NO: 58, 41 and 146, respectively; Preferably, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 34, 148 and 149, SEQ ID NO: 37, 150 and 151, or SEQ ID NO: 40, 152 and 149, respectively; 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 71, 153 and 154, SEQ ID NOs: 104, 155 and 156, or SEQ ID NOs: 107, 157 and 154, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the CDR1, CDR2 and / or CDR3 comprise an amino acid sequence in which one, two or three mutations occur in the HCDR1, HCDR2 and / or HCDR3 of claim 1, the mutations being selected from insertions, deletions and / or substitutions, and the substitutions are preferably conservative amino acid substitutions.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the CDR1, CDR2 and / or CDR3 comprise an amino acid sequence having at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the HCDR1, HCDR2 and / or HCDR3.

5. The antibody or antigen-binding fragment thereof comprises a single domain antibody comprising the CDR1, CDR2 and CDR3 of claim 1, Preferably, the single domain antibody comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101; Optionally, said single domain antibody comprises an amino acid sequence in which at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation occurs compared to the amino acid sequence set forth in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101, said mutations being selected from insertions, deletions and / or substitutions, said substitutions being preferably conservative amino acid substitutions; Optionally, the single domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101.

6. The antibody comprises a FR region in a VHH domain shown in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31, and 92-101; Optionally, the antibody comprises an amino acid sequence in which at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation occurs compared to the FR region of the VHH domain as set forth in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101, the mutations being selected from insertions, deletions and / or substitutions, the substitutions being preferably conservative amino acid substitutions; Optionally, the antibody or antigen-binding fragment thereof of claim 1 comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a FR region in a VHH domain set forth in any one of SEQ ID NOs: 27, 7-21, 24-26, 28-31 and 92-101.

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is selected from (1) a chimeric antibody or fragment thereof, (2) a humanized antibody or fragment thereof, or (3) a fully human antibody or fragment thereof.

8. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises or does not comprise an antibody heavy chain constant region, optionally the antibody heavy chain constant region is selected from human, alpaca, mouse, rat, rabbit and sheep, optionally the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE and IgD, optionally the IgG is selected from IgG1, IgG2, IgG3 and IgG4, optionally the heavy chain constant region is selected from an Fc region, a CH3 region and a complete heavy chain constant region, preferably the heavy chain constant region is a human Fc region, and preferably the antibody or antigen-binding fragment thereof is a heavy chain antibody.

9. The antibody or antigen-binding fragment thereof of claim 1, further comprising a therapeutic agent or tracer coupled to said antibody or antigen-binding fragment thereof, wherein preferably said therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulatory agent, and said tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

10. The antibody or antigen-binding fragment thereof of claim 1, further comprising another functional molecule linked to the antibody or antigen-binding fragment thereof, preferably the other functional molecule being selected from one or more of a signal peptide, a protein tag, a cytokine, an angiogenesis inhibitor, and an immune checkpoint inhibitor.

11. A multispecific antigen-binding molecule, comprising the antibody or antigen-binding fragment thereof of claim 1 and an antigen-binding molecule that binds to an antigen other than BCMA, or that binds to a different BCMA epitope than the antibody or antigen-binding fragment thereof of claim 1, optionally wherein the antigen other than BCMA is selected from CD3 (preferably CD3ε), CD16, CD137, CD258, PD-1, PD-L1, 4-1BB, CD40, CD64, EGFR, VEGF, HER2, HER1, HER3, IGF-1R, phosphatidylserine (PS), C-Met, HSA, GPRC5D, MSLN, blood-brain barrier receptors, GPC3, PSMA, CD33, GD2, ROR1, ROR2, FRα, and Gucy2C; Preferably, the antigen-binding molecule is an antibody or an antigen-binding fragment thereof, Preferably, the multispecific antigen-binding molecule is bispecific, trispecific or tetraspecific; Preferably, the multispecific antigen-binding molecule is bivalent, trivalent, tetravalent, pentavalent or hexavalent.

12. 12. An isolated nucleic acid fragment, said nucleic acid fragment encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to 10 or a multispecific antigen-binding molecule of claim 11.

13. A vector comprising the nucleic acid fragment of claim 12.

14. 13. A host cell, said host cell comprising the nucleic acid fragment according to claim 12, preferably said cell being a prokaryotic or eukaryotic cell, such as a bacterial (e.g. E. coli), a fungal (e.g. yeast), an insect cell or a mammalian cell (e.g. a CHO cell line or a 293T cell line).

15. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the multispecific antigen-binding molecule of claim 11, the method comprising culturing a cell comprising a nucleic acid fragment encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the multispecific antigen-binding molecule of claim 11, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, the multispecific antigen-binding molecule of claim 11, or a nucleic acid fragment encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 10 or the multispecific antigen-binding molecule of claim 11, optionally further comprising a pharma- ceutical acceptable carrier, diluent or excipient, and optionally further comprising an additional anti-tumor agent.

17. 17. The pharmaceutical composition according to claim 16 for treating a tumor or cancer. Optionally, the tumor or cancer is a BCMA-expressing tumor or cancer, preferably, the tumor or cancer is a B cell lymphoma, more preferably, multiple myeloma (MM).

18. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the multispecific antigen-binding molecule according to claim 11, or a nucleic acid fragment encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the multispecific antigen-binding molecule according to claim 11.

19. A method for detecting BCMA expression in a biological sample, the method comprising contacting the biological sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof and BCMA, preferably the method further comprising detecting formation of the complex to indicate the presence or expression level of BCMA in the sample.

Images