Antibody binding to BCMA and multispecific antibody comprising same

Abstract

A monoclonal antibody specifically binding to BCMA, a BCMA- and CD3-targeted bispecific antibody constructed on the basis of said monoclonal antibody, and a BCMA / GPRC5D / CD3-targeted trispecific antibody. Also provided are a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell and a method. Also provided are an immunoconjugate, a bispecific molecule, a trispecific molecule and a pharmaceutical composition comprising the antibody, and a diagnosis and treatment method using the anti-BCMA antibody, the BCMA / CD3-targeted bispecific antibody or the BCMA / GPRC5D / CD3-targeted trispecific antibody.

Description

Antibodies that bind to BCMA and multispecific antibodies containing the same Technical Field

[0001] The present invention provides a monoclonal antibody that specifically binds to BCMA, a bispecific antibody for BCMA and CD3 constructed based on the BCMA monoclonal antibody, and a trispecific antibody for BCMA / GPRC5D / CD3. Also provided are nucleic acid molecules encoding the antibodies, expression vectors, host cells, and methods for expressing the antibodies. The present invention also provides immunoconjugates, bispecific molecules, trispecific molecules, and pharmaceutical compositions comprising the antibodies, as well as diagnostic and therapeutic methods using the anti-BCMA antibodies, BCMA / CD3 bispecific antibodies, and BCMA / GPRC5D / CD3 trispecific antibodies of the present invention. Background Art

[0002] B cell maturation antigen (BCMA, CD269) is a member of the TNF receptor superfamily. The expression of BCMA is restricted to the B cell lineage, where it is mainly expressed in the interfollicular zone of the germinal center and on differentiated plasma cells and plasmablasts. BCMA binds to two distinct ligands, proliferation-inducing ligand (APRIL) and B cell activating factor (BAFF, also known as BlyS, TALL-1 and THANK). The ligand of BCMA binds to two additional TNF receptors, transmembrane activator-calcium regulator-cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R, also known as BR3). TACI binds to APRIL and BAFF, while BAFF-R shows limited but high-affinity binding to BAFF. Taken together, BCMA, TACI, BAFF-R and their corresponding ligands regulate different aspects of humoral immunity, B cell development and homeostasis. BCMA is almost absent on naive and memory B cells (Novak et al., Blood, 103, 689-94 (2004)), but is selectively induced during plasma cell differentiation, during which it can support humoral immunity by promoting the survival of normal plasma cells and plasmablasts (O'Conner et al., J. Exp Med., 199, 91-98 (2004)). BCMA has been reported to be expressed in primary multiple myeloma (MM) samples.

[0003] G protein-coupled receptor family C group 5 member D (GPRC5D), an orphan receptor, was found to be highly expressed in plasma cells of multiple myeloma patients and was associated with the patients' survival rate (Atamaniuk, J., et al. (2012). "Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma." Eur J Clin Invest 42(9):953-960.). Further studies have shown that GPRC5D is expressed on the surface of plasma cells in normal human blood cells, while GPRC5D expression on the surface of other blood cells is negative. GPRC5D is highly expressed on the surface of CD38+CD138+ plasma cells isolated from multiple myeloma patients (Kodama, T., et al. (2019). "Anti-GPRC5D / CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma." Mol Cancer Ther 18(9): 1555-1564.). Analysis of patient multiple myeloma cells found that there was no correlation between GPRC5D and BCMA expression (Smith, EL, et al. (2019). "GPRC5Dis a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells." Sci Transl Med 11(485).)(Pillarisetti, K., et al. (2020). "A T-cell-redirecting bispecific G-protein-coupled receptor class 5member D x CD3 antibody to treat multiple myeloma." Blood 135(15):1232-1243.), so GPRC5D can be used as a tumor-specific target for multiple myeloma. In addition, GPRC5D can also be used as a tumor-specific target for other tumors.

[0004] CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by the dimerization of two of the following four different chains: ε, ζ, δ, and γ. CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. Antibodies against CD3 have been shown to aggregate CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of TCRs by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. In addition, it has been proposed that bispecific antibodies capable of binding to CD3 and targeting tumor surface antigens can connect tumor cells and T cells, thereby directly activating T cells and releasing granzymes, perforins, and cytokines to kill tumors, thereby achieving the therapeutic purpose of suppressing tumors.

[0005] T cell-based antibody therapy has played an important role in combating a variety of cancers, including multiple myeloma (MM). Bispecific antibodies (BsAbs) are based on a dual-targeting antibody design that uses one arm to bind to the relevant antigen on the tumor cell and the other arm to bind to the CD3 co-receptor complex on the T cell, redirecting the cytotoxic T cells and gathering them near the tumor cells to achieve tumor cell dissolution. The ability of BsAbs to dissolve tumors depends on the expression distribution and abundance of tumor-associated antigens on tumor cells. This leads to the selectivity and treatment limitations of BsAbs therapy for patients. Therefore, due to the downregulation of the expression of tumor-associated antigens, the immune selection pressure decreases, leading to immune escape and thus disease recurrence.

[0006] Since BCMA and GPRC5D are both specific tumor-associated antigens on the surface of multiple myeloma (MM) tumor cells, the degree of tumor cell heterogeneity in MM patients is high, and the expression of BCMA and GPRC5D on MM cells is relatively independent. BCMA-positive or GPRC5D-positive MM cells exist in patients. If treated with BCMAxCD3 BsAb or GPRC5DxCD3 BsAb, it cannot completely kill BCMA-negative or GPRC5D-negative MM cells, eventually leading to disease recurrence.

[0007] Although existing technologies include antibodies targeting BCMA and bispecific or multispecific antibodies constructed based on BCMA antibodies or fragments, such as Teclistamab (US2017 / 0051068A1), which are highly safe and provide deep and lasting remissions to MM patients who have failed multiple lines of treatment and have entered Phase III clinical trials, other BCMA / CD3 bispecific antibodies such as AMG420 and REGN5458 are also in clinical research. However, known antibodies still have areas that need improvement. For example, these antibodies have weak T cell killing effects and high cytokine release levels, or have low affinity for cells expressing human BCMA, or cannot effectively eliminate tumor cell lines with low human BCMA expression, or specifically induce T cells to have nonspecific killing effects on BCMA-negative cells, or BCMA expression mutations and downregulation caused by treatment.

[0008] Therefore, there is a need for a new BCMA antibody and an anti-GPRC5D / BCMA / CD3 trispecific antibody constructed based on it, which can overcome the above shortcomings and induce optimal T cell killing and low factor release levels.

[0009] Summary of the Invention

[0010] The present invention first provides a novel anti-BCMA antibody, such as a VHH antibody or an antibody comprising VHH, such as VHH-Fc, and a bispecific antibody constructed using the same and CD3 antibody.

[0011] In some embodiments, the anti-BCMA antibodies specifically bind to BCMA (e.g., human BCMA) with high affinity. In some embodiments, the anti-BCMA antibodies bind to cells expressing BCMA, such as tumor cells that endogenously express BCMA.

[0012] The anti-CD3 / BCMA bispecific antibodies of the present invention have one or more of the following characteristics:

[0013] (1) binding to human BCMA, for example, binding to human BCMA expressed on cells, for example, binding to BCMA with high affinity;

[0014] (2) specifically induce T cells to kill BCMA-positive tumor cells and / or have no (e.g., significant) nonspecific killing effect on BCMA-negative cells;

[0015] (3) having good cytokine induction specificity; for example, inducing cells (e.g., T cells) to release cytokines, such as interferons such as IFNγ; tumor necrosis factors such as TNFα and / or interleukins such as IL-6, only in cells expressing BCMA (e.g., BCMA-positive tumor cells);

[0016] (4) The trend of induced cytokine release is consistent with its killing effect on BCMA-positive cells, for example, the stronger the killing effect, the higher the factor release;

[0017] (5) Effective treatment of BCMA-related indications.

[0018] The present invention also provides a novel anti-GPRC5D antibody, such as a VHH antibody or an antibody comprising VHH, such as VHH-Fc, and a multispecific antibody constructed using the same, together with a BCMA antibody and a CD3 antibody.

[0019] The present invention also provides a multispecific antibody that simultaneously targets BCMA and GPRC5D, which can cover both BCMA-positive patients and GPRC5D-positive patients, while also avoiding immune escape and disease recurrence caused by down-regulation or non-expression of a single target, thereby improving the therapeutic effect and bringing clinical benefits.

[0020] Therefore, the present invention constructed a trispecific antibody of BCMA / GPRC5D / CD3. By adjusting the position of the CD3 antibody and the position of the BCMA / GPRC5D antibody, various combinations were performed to screen for trispecific antibodies with optimal induction of T cell killing and low factor release levels.

[0021] In some embodiments, the anti-BCMA / GPRC5D / CD3 trispecific antibodies of the present invention have one or more of the following properties:

[0022] (1) specifically binds to human BCMA, human GPRC5D, and human CD3, for example, binds to proteins on the surface of cells expressing human BCMA and human GPRC5D proteins (e.g., tumor cells endogenously expressing human BCMA and human GPRC5D), for example, it does not bind to other GPRC family members other than GPRC5D, for example, it does not bind to GPRC5A, GPRC5B, and GPRC5C, or binds to them very weakly;

[0023] (2) specifically induce T cells to kill BCMA and / or GPRC5D-positive tumor cells;

[0024] (3) specifically activates T cells (e.g., as indicated by increased expression of CD25 and / or CD69), e.g., better than known control antibodies, and promotes T cell apoptosis (e.g., as indicated by increased expression of PD1 and / or TIM3);

[0025] (4) having good cytokine induction specificity; for example, inducing cells (e.g., T cells) to release cytokines, such as interferons, such as IFNγ; tumor necrosis factors, such as TNFα and / or interleukins, such as IL-6, only in cells expressing human BCMA and / or human GPRC5D (e.g., BCMA and / or GPRC5D-positive tumor cells);

[0026] (4) The trend of induced cytokine release was consistent with its killing effect on cells expressing human BCMA and / or GPRC5D (BCMA-positive and / or GPRC5D-positive), for example, the stronger the killing effect, the higher the factor release;

[0027] (5) specifically induce T cells to kill tumor cells expressing human BCMA and / or GPRC5D (BCMA-positive and / or GPRC5D-positive) (e.g., the killing activity is stronger than the control), and there is no nonspecific killing of BCMA- and GPRC5D-negative cells;

[0028] (6) No (e.g., significant) effect on promoting T cell factor release in BCMA- and GPRC5D-negative cells;

[0029] (7) specifically binds to tumor cells expressing human BCMA and / or GPRC5D (BCMA-positive and / or GPRC5D-positive), and the binding activity is stronger than that of the control;

[0030] (8) Treating tumors in vivo, such as inhibiting tumor growth and / or not affecting the body weight of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows the structure of a bispecific antibody.

[0032] FIG2 shows the binding of anti-human BCMA chimeric antibodies to cell lines expressing human BCMA protein.

[0033] FIG3 shows the binding of anti-human BCMA humanized antibodies to tumor cell lines expressing human BCMA protein.

[0034] FIG4 shows the binding of bispecific antibodies to cell lines expressing human BCMA protein.

[0035] FIG5 shows the specific killing of tumor cell lines expressing human BCMA protein by bispecific antibodies.

[0036] FIG6 shows the release of factors in the killing system of bispecific antibodies against tumor cell lines expressing human BCMA protein.

[0037] Figure 7 shows a diagram of the trispecific antibody structure.

[0038] FIG8 shows the binding of trispecific antibodies to tumor cell lines expressing human BCMA protein.

[0039] FIG9 shows the killing effect of trispecific antibodies on tumor cell lines expressing human BCMA protein.

[0040] FIG10 shows the detection results of T cell activation and exhaustion markers in the killing system of tumor cell lines expressing human BCMA protein by trispecific antibodies.

[0041] FIG11 shows the release of factors in the killing system of tumor cell lines expressing human BCMA protein by trispecific antibodies.

[0042] FIG12 shows the killing results of the trispecific antibody and the control bispecific antibody alone or in combination against the multiple myeloma tumor cell line MOLP8.

[0043] FIG13 shows the release of TNFα in the killing system of multiple myeloma cell line MOLP8 by the trispecific antibody and the control bispecific antibody alone or in combination.

[0044] FIG14 shows the binding of the trispecific antibody, control bispecific antibody, and trispecific antibody to multiple myeloma tumor cell lines ARD and AMO-1.

[0045] FIG15 shows the killing results of the trispecific antibody, the control bispecific antibody and the triple antibody against the multiple myeloma tumor cell line ARD.

[0046] FIG16 shows the killing results of the trispecific antibody, the control bispecific antibody and the triple antibody against the multiple myeloma tumor cell line AMO-1.

[0047] FIG17 shows the release of TNFα in the killing system of multiple myeloma tumor cell line ARD by trispecific antibodies, control bispecific antibodies and triple antibodies.

[0048] FIG18 shows the release of TNFα in the killing system of the multiple myeloma tumor cell line AMO-1 by the trispecific antibody, the control bispecific antibody and the triple antibody.

[0049] FIG19 shows the tumor inhibitory effect of the trispecific antibody in the NCI-H929 tumor-bearing humanized mouse model.

[0050] FIG20 shows the tumor inhibitory effect of the trispecific antibody in a MOLP8 tumor-bearing humanized mouse model.

[0051] FIG21 shows the detection of camel-derived anti-GPRC5D VHH antibodies binding to human GPRC5D protein.

[0052] FIG22 shows the detection of camel-derived anti-GPRC5D VHH antibodies binding to cynomolgus monkey GPRC5D protein.

[0053] FIG23 shows the detection of humanized anti-GPRC5D VHH-Fc antibody binding to GPRC5D.

[0054] Figure 24 shows the detection of humanized anti-GPRC5D VHH antibodies binding to GPRC5A.

[0055] Figure 25 shows the detection of humanized anti-GPRC5D VHH antibodies binding to GPRC5B.

[0056] Figure 26 shows the detection of humanized anti-GPRC5D VHH antibodies binding to GPRC5C.

[0057] Detailed Description of the Invention

[0058] It should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0059] I. Definition

[0060] For the purpose of interpreting this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0062] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0063] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0064] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0065] As used herein, the term "BCMA" refers to a tumor-associated antigen B cell maturation antigen, also known as BCMA, TR17_human, TNFRSF17 (e.g., human BCMA protein under accession number UniProt Q02223). In one embodiment, the human BCMA protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 77 (or its sequence without a human Fc tag), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consists of said sequence. In one embodiment, the antigen-binding region that binds to BCMA in the anti-BCMA antibody, or antigen-binding fragment thereof, or trispecific antibody of the present invention has high affinity binding activity to cells expressing human BCMA, for example, having a higher binding affinity than a control antibody (e.g., 269B094). In one embodiment, the binding is determined by flow cytometry.

[0066] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR), i.e., the T cell engaging antigen, the T cell surface glycoprotein CD3, which is composed of a homodimer or heterodimer formed by two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (hCD3ε) comprises the amino acid sequence set forth in UniProtKB / Swiss-Prot: P07766. Human CD3-δ (hCD3δ) comprises the amino acid sequence set forth in UniProtKB / Swiss-Prot: P04234. In some embodiments, the CD3 described herein refers to CD3 from humans or monkeys (e.g., cynomolgus monkeys).

[0067] As used herein, the term "antibody that binds to CD3" or "anti-CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to a single CD3 subunit (e.g., ε, δ, γ, or ζ), as well as antibodies and antigen-binding fragments thereof that specifically recognize or bind to a dimeric complex of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) and bind thereto. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3, and / or CD3 expressed on the cell surface. Soluble CD3 includes natural CD3 protein and recombinant CD3 protein variants, for example, monomeric and dimeric CD3 structures that lack a transmembrane region or are otherwise not bound to the cell membrane. In one embodiment, the antigen-binding region that binds to CD3 in the multispecific antibody of the present invention may have low binding activity to CD3 or cells expressing CD3 (e.g., T cells). The binding affinity of the antibody to CD3 can be detected by flow cytometry or biofilm optical interferometry. Preferably, in the trispecific antibody molecule of the present invention, the antigen-binding region that binds CD3 has a binding affinity for human or monkey (cynomolgus monkey) CD3 of between 1-1000 nM. In some embodiments, the antigen-binding region that binds CD3 in the trispecific antibody of the present invention binds to human and / or monkey (e.g., cynomolgus monkey) CD3 with a lower binding affinity, thereby being able to activate human and / or monkey (e.g., cynomolgus monkey) T cells.

[0068] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells can also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).

[0069] The term "GPRC5D" refers to a tumor-associated antigen G protein-coupled receptor family C group 5 member D (e.g., human GPRC5D protein under accession number NP_061124.1 or cynomolgus monkey GPRC5D protein under XP_005570249.2). In one embodiment, the human GPRC5D protein of the present invention comprises, or consists of, the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In one embodiment, the antigen-binding region that binds to GPRC5D in the trispecific antibody of the present invention has high-affinity binding activity for cells expressing human GPRC5D, for example, having a higher binding affinity than a control antibody (e.g., GC5B596). In one embodiment, the binding is determined by flow cytometry. In one embodiment, the antigen-binding region in the trispecific antibody of the present invention that binds to GPRC5D is cross-reactive to human and monkey (eg, cynomolgus monkey) GPRC5D, ie, it can bind to human and monkey (eg, cynomolgus monkey) GPRC5D.

[0070] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. 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 can be further divided into hypervariable regions (complementarity determining regions (CDRs) interspersed with relatively conserved regions (framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions. In some embodiments, the heavy chain constant region HC of the antibody of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. In some embodiments, the heavy chain constant region comprises a LALA mutation. In some embodiments, the heavy chain constant region comprises D265A and P329A mutations. In some embodiments, the heavy chain constant region comprises a LALA mutation and D265A and P329A mutations. In some embodiments, the heavy chain constant region of the multispecific antibody molecule of the present invention comprises a "knob into hole mutation". In some preferred embodiments, the heavy chain constant region HC of the antibody of the present invention

[0071] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 89 or 92;

[0072] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 89 or 92; or

[0073] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 89 or 92.

[0074] In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region.

[0075] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 91 or 93;

[0076] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 91 or 93; or

[0077] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 91 or 93.

[0078] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0079] The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody that has fewer amino acid residues than the intact or complete antibody and that is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, and single-domain antibodies (sdAbs). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. For example, Fab fragments can be obtained by digesting an intact antibody with papain. In addition, digesting an intact antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab', a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, edited by W.E. Paul, Raven Press, NY (1993)). The Fv fragment consists of the VL and VH domains of a single antibody arm. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, recombinant methods can be used to connect them via a synthetic linker peptide that enables the two domains to be produced as a single protein chain, in which the VL and VH regions are paired to form a single-chain Fv (scFv). The antibody fragment can be obtained by chemical methods, recombinant DNA methods, or protease digestion.

[0080] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0081] The terms "VHH" or "VHH antibody" are used interchangeably herein and generally refer to an antibody that comprises or consists of only one heavy chain variable region and has antigen binding activity. VHHs generally comprise three CDRs in four highly conserved framework regions and generally have the following structure: FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; CDR1 to CDR3 refer to complementarity determining regions 1-3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, and preferably the boundaries of the three CDRs in the variable region sequence can be defined by IMGT. VHHs generally include only heavy chain variable domains derived from heavy chain antibodies lacking light chains, also known as nanobodies. The VHHs used in the present invention are preferably from camelids, such as alpacas, or are humanized or sequence-optimized forms thereof (e.g., affinity mature forms to increase binding affinity). In some embodiments, a VHH of the invention is a monovalent, monospecific polypeptide molecule that consists of, or consists essentially of, a single heavy chain variable region (eg, the heavy chain variable region of a heavy chain antibody).

[0082] The single domain antibodies or VHHs of the present invention may also be contained in a larger polypeptide / protein. Examples of polypeptides / proteins containing the VHHs of the present invention include, but are not limited to, heavy chain antibodies (HcAbs) or multispecific antibodies or fusion proteins.

[0083] The "heavy chain antibody" described in the present invention refers to an antibody without a light chain, for example, its N-segment to C-segment may include VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3, or may include VH-CH1-CH2-CH3. The heavy chain antibody of the present invention may also encompass homodimers, such as heavy chain dimer antibodies without light chains. The heavy chain antibody may include VH from a standard antibody or VH from a single domain antibody. For example, the VH in a heavy chain antibody may be VHH. In some embodiments, the heavy chain antibody of the present invention may be a heavy chain antibody having a framework region and / or a heavy chain constant region derived from a camelid (llama, camel, especially alpaca), a humanized form thereof or a sequence-optimized form thereof (affinity matured form), or a fragment thereof (e.g., a fragment comprising at least a portion of the constant region). The heavy chain antibody of the present invention also encompasses an antibody formed by fusing a heavy chain variable region or VHH with an Fc region (e.g., a human IgG Fc region, such as a human IgG1 or IgG4 Fc region). When "VHH" is mentioned in the context of a heavy chain antibody or a multispecific antibody or a fusion protein, it should be understood that it is part of a multispecific antibody and not as a separate molecule.

[0084] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor.

[0085] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Additionally, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule.

[0086] As used herein, the term "target binding region" refers to the portion of a multispecific binding molecule, such as a bispecific binding molecule, that binds to a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the remainder of the multispecific antibody molecule and may bind or not bind to its target as a separate entity. The target binding region can also be a receptor or ligand, or a domain of a receptor that is capable of binding to a ligand. In the case of multispecific antibodies, the "target binding region" is also referred to as an "antigen binding region." In one embodiment, the antigen binding region for the multispecific antibody molecule of the present invention comprises a VH / VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), the VH / VL pair being comprised in a single polypeptide chain (e.g., in an scFv) or in two separated polypeptide chains (e.g., in a Fab heavy chain and a Fab light chain, respectively). In one embodiment, the antigen binding region for the multispecific antibody molecule of the present invention can be a Fab. In one embodiment, the antigen binding region of a multispecific antibody molecule for use in the invention may comprise only VH, eg, be derived from VHH, eg, be VHH.

[0087] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding regions, each of which binds to the same epitope of the same antigen. For example, the present invention provides a monospecific antibody against BCMA.

[0088] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen binding regions, each of which antigen binding sites binds to different epitopes of the same antigen or to different epitopes of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein are bispecific antibodies having binding specificity for a first antigen and a second antigen. For example, the present invention provides bispecific antibodies for BCMA and CD3. In one embodiment, provided herein are trispecific antibodies having binding specificity for a first antigen, a second antigen, and a third antigen. For example, the present invention provides trispecific antibodies for BCMA, GPRC5D, and CD3.

[0089] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target binding region and a second target binding region, wherein the first target binding region binds to one target and the second target binding region binds to another target. Thus, the multispecific binding molecules according to the present invention comprise specificity for at least two different targets. The molecules according to the present invention also encompass multispecific molecules comprising multiple target binding regions, such as trispecific binding molecules. In some embodiments, where the binding molecule is an antibody, the target is an antigen. In some embodiments, the multispecific binding molecules of the present invention are bispecific antibodies or trispecific antibodies.

[0090] When referring to a "first antigen-binding region" in a bispecific antibody or a trispecific antibody, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody may contain one or more first antigen-binding regions. For example, a trispecific antibody contains a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region, but may contain one or more first antigen-binding regions, one or more second antigen-binding regions, and one or more third antigen-binding regions. In some embodiments, the bispecific antibody molecule of the present invention comprises at least one antigen-binding region that specifically binds to CD3 and at least one antigen-binding region that specifically binds to BCMA. In some embodiments, the bispecific antibody molecule of the present invention comprises one antigen-binding region that specifically binds to CD3 and one or two antigen-binding regions that specifically bind to BCMA. In some embodiments, the trispecific antibody molecule of the present invention comprises at least one antigen-binding region that specifically binds to GPRC5D, at least one antigen-binding region that specifically binds to BCMA, and at least one antigen-binding region that specifically binds to CD3. In some embodiments, the trispecific antibody molecule of the present invention comprises an antigen binding region that specifically binds to GPRC5D, an antigen binding region that specifically binds to BCMA, and an antigen binding region that specifically binds to CD3.

[0091] When referring to "the antigen-binding region is derived from an antibody", it means that the binding domain constituting the antigen-binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, a fragment of the antigen-binding region that specifically binds to the antigen, such as Fab, is or is derived from a corresponding fragment of the antibody, such as Fab, or the heavy chain variable region and / or light chain variable region of the antigen-binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen-binding region are CDRs of the antibody. The term "derived from" means that the fragment in the antigen-binding region is substantially the same as the fragment of the antibody from which it is derived, but has a mutation at one or more sites, such as a substitution, deletion or addition. In a specific embodiment, the mutation is not in the CDR of the antibody. In a specific embodiment, the mutation is not in the variable region of the antibody.

[0092] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions.

[0093] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0094] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0095] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position of the Kabat numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0096] In one embodiment, the HCDRs in the VHH antibodies of the invention are determined according to the IMGT protocol.

[0097] The term "Fc domain" or "Fc region" or "Fc fragment" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD classes of antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE classes of antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. As used herein, the term "Fc domain" or "Fc region" or "Fc fragment" does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include part of the hinge region at the N-terminus of the heavy chain constant region, such as EPKSS or EPKSC. In some embodiments, the heavy chain constant region Fc suitable for the present invention is derived from an antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3 or IgG4, preferably the constant region of IgG1. In some embodiments, the Fc region comprises a mutation that reduces binding to Fcγ receptors, such as a LALA mutation, a D265A mutation and / or a P329A mutation, preferably comprising a LALA mutation and D265A and P329A mutations. In some embodiments, Fc comprises an amino acid sequence as shown in SEQ ID NO: 80, 90, 97 or SEQ ID NO: 98, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of said amino acid sequence. In some embodiments of the present invention, Fc fragments dimerize to form Fc dimers. In some embodiments, Fc fragments heterodimerize to form Fc heterodimers. In the case where the Fc fragment heterodimerizes into a heterodimer, the Fc fragment may comprise a mutation for heterodimerization, such as a knob into hole mutation.

[0098] Examples of "effector functions" of immunoglobulins include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B-cell receptor), and B-cell activation.

[0099] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0100] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a camelid VHH antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing portions of the variable region that do not participate in binding with corresponding portions of a human antibody).

[0101] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0102] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0103] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes or be conservatively modified variants.

[0104] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.

[0105] "Knob-in-hole" mutations or "knob-in-knob" mutations are used herein to refer to the introduction of mutations into a first Fc polypeptide and a second Fc polypeptide, respectively, using the "knob-in-knob" technique to form a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and at the interface of the second Fc polypeptide. It is known in the art that the "knob-in-hole" technique can be used to modify the interface between different chains of an antibody molecule to promote the correct association of the chains of the antibody molecule. Typically, the technique involves introducing a "protrusion / knob" at the interface of one chain and a corresponding "hole / knob" at the interface of the other chain to be paired with it, so that the protrusion can be placed in the cavity. A preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired with it. The protrusion can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Another optional interface is the CL domain of the light chain and the CH1 domain of the heavy chain of the Fab fragment described above, which promotes correct heterodimerization between the two chains of the Fab fragment by establishing a protrusion-cavity interaction.

[0106] In this article, the antibody constant region or antibody constant domain, including CH1, CL and Fc domains and CH2, CH3 and optional CH4 domains constituting the Fc domain, can be selected according to the intended function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG or IgM region, in particular an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. The immunoglobulin constant region can have a native sequence or a variant sequence.

[0107] As used herein, the term "joint" refers to any molecule that enables the direct connection of the different parts of a bispecific binding molecule. The example of a covalently linked joint between different molecular parts includes a peptide joint and a non-protein polymer, including but not limited to a copolymer of polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol. In some embodiments, the joint is a peptide joint (also referred to as a "connecting peptide"), which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or a hinge region from an immunoglobulin, for connecting the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule. For example, a peptide joint can connect the first target binding region of the binding molecule to the second target binding region. For example, a peptide joint can also connect a part of an antibody to another part of the antibody, such as connecting a light chain variable region to a heavy chain variable region. Preferably, the peptide joint has a length that is sufficient to connect two entities in a manner such that they maintain their conformations relative to each other so as not to hinder the desired activity. In one embodiment, the connecting peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (GS)n, (GGS)n, (GGSGG)n, (GSGGS)n, (GGGGS)n, (GGGS)n and (GGGGS)nG, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers and other flexible linkers. In some embodiments, the peptide linker is (GGGGS)n, wherein n=1, 2, 3 or 4, for example, the sequence shown in SEQ ID NO:95, 99 or 100. In some embodiments, the peptide linker is the sequence shown in SEQ ID NO:101.

[0108] In yet another embodiment, the connecting peptide is a hinge region or a portion of a hinge region from an immunoglobulin, including a native hinge region or a portion thereof, or a mutated hinge region or a portion thereof. In one embodiment, the connecting peptide is, for example, a hinge region or a portion thereof (e.g., EPKSC) of an immunoglobulin (e.g., IgG, e.g., IgG1, IgG2, IgG3, or IgG4), or a mutated hinge region or a portion thereof, e.g., EPKSS. Alternatively, a computer program can be used to simulate the three-dimensional structure of proteins and peptides, or by phage display methods, to rationally design suitable flexible connecting peptides.

[0109] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0110] The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences that are operatively linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which recombinant polynucleotides are incorporated.

[0111] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0112] The term "treat," ...

[0113] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0114] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (eg, immunosuppressants).

[0115] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0116] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0117] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0118] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0119] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0120] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0121] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0122] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein. The term "tumor" encompasses both solid tumors and hematologic tumors.

[0123] The term "anti-tumor effect" or "tumor inhibitory effect" or "tumor suppressive effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a decrease in tumor volume, a decrease in tumor cell number, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0124] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0125] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0126] The term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody or multispecific antibody of the present invention, and (ii) other therapeutic agents) are administered to a patient as separate entities simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0127] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0128] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. In some embodiments, the tissue sample is tumor tissue. The tissue sample may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0129] II. Anti-BCMA Antibodies

[0130] In one aspect, the present invention provides a BCMA antibody having a higher binding affinity to BCMA. In some embodiments, the BCMA antibody of the present invention is suitable for constructing an antigen-binding region in a multispecific antibody molecule.

[0131] In some embodiments, the anti-BCMA antibodies or antigen-binding fragments thereof of the present invention bind to BCMA (e.g., human BCMA) with higher affinity. In some embodiments, the anti-BCMA antibodies or antigen-binding fragments thereof of the present invention can bind to human BCMA with high affinity, e.g., their K D The value is less than or equal to about 0.5 nM, such as less than or equal to about 5, 4, 3, 2, or 1 pM.

[0132] In some embodiments, the anti-BCMA antibodies or antigen-binding fragments thereof of the present invention bind to BCMA expressed on cells. In some embodiments, the affinity of the anti-BCMA antibodies to BCMA expressed on cells is determined by flow cytometry.

[0133] Single domain antibodies

[0134] In some embodiments, the anti-BCMA antibodies of the present invention are single domain antibodies, particularly VHH antibodies.

[0135] Single-domain antibodies, or VHH antibodies, have a molecular weight approximately one-tenth that of a human IgG molecule and a physical diameter of only a few nanometers. Due to their small molecular size, single-domain antibodies offer advantages over conventional four-chain antibodies: high stability and solubility, as well as the ability to recognize hidden antigenic sites. Furthermore, single-domain antibodies are cheaper to prepare than conventional four-chain antibodies. In addition to their use as individual molecules, single-domain antibodies are also suitable components for constructing multispecific molecules.

[0136] In some embodiments, the anti-BCMA single-domain antibody of the present invention is a VHH antibody comprising or consisting of a heavy chain variable region, which generally has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be determined according to any CDR definition scheme described in the "Definitions" section. Preferably, the boundaries of the three CDRs in the VHH sequence can be defined using IMGT.

[0137] In some embodiments, the anti-BCMA VHH antibody of the present invention comprises

[0138] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23, or

[0139] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0140] Preferably, the CDR sequences are defined according to IMGT.

[0141] In some embodiments, the anti-BCMA VHH antibody of the present invention comprises or consists of a heavy chain variable region comprising

[0142] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23, or

[0143] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0144] Preferably, the CDR sequences are defined according to IMGT.

[0145] In some embodiments, the anti-BCMA VHH antibodies of the present invention comprise complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-BCMA VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0146] In some embodiments, the VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 1, 5, or 19, or the VHH CDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 1, 5, or 19.

[0147] In some embodiments, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 6, 9, 13 or 17, or the VHH CDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2, 6, 9, 13 or 17.

[0148] In some embodiments, the VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 3, 7 or 20, or the VHH CDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 3, 7 or 20.

[0149] In one embodiment, the anti-BCMA VHH antibody of the present invention comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0150] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 9 or 13, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3;

[0151] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 5, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 6, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7;

[0152] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

[0153] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0154] In one embodiment, an anti-BCMA VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein said heavy chain variable region comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein

[0155] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 9 or 13, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3;

[0156] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 5, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 6, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7;

[0157] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

[0158] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0159] In some embodiments, the anti-BCMA VHH antibodies of the present invention comprise or consist of a heavy chain variable region, wherein the heavy chain variable region

[0160] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23;

[0161] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23; or

[0162] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22, or 23, preferably, the amino acid changes do not occur in the CDR regions.

[0163] In some embodiments, the anti-BCMA VHH antibody of the present invention comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22, or 23.

[0164] In some embodiments, the VHH antibodies of the present invention comprise CDR amino acid sequences and / or framework (FR) amino acid sequences derived from camelid heavy chain antibodies produced by immunizing camelids (e.g., alpacas). In some embodiments, the VHH monoclonal antibodies of the present invention derived from camelid heavy chain antibodies can be engineered, for example, to comprise framework region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelid mammalian species. In one embodiment, to further improve the properties (e.g., affinity) of the engineered antibodies, camelid amino acid residues located at corresponding positions in the parent camelid antibody can be introduced into the engineered antibodies at one or more positions (e.g., framework regions) by back mutation.

[0165] In one embodiment, the VHH antibody of the present invention is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, especially framework region sequences, in a non-human natural VHH sequence (e.g., a VHH sequence from camelids or alpacas immunization) with residues at corresponding positions in the heavy chain VH of a conventional human antibody. Methods for humanizing VHHs are well known in the art, such as the method described in Example 3. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of single-domain antibodies. Tests for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0166] In some embodiments, the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:

[0167] Determine the CDR loop structure of a parent single domain antibody (e.g., a camelid VHH antibody screened from a phage display library);

[0168] Find the closest homologous sequence for each V / J region in the human germline sequence database as a template, for example, by aligning the IMGT human antibody heavy chain variable region germline gene database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) and selecting a heavy chain variable region germline gene with high homology to the VHH antibody as a template;

[0169] The CDRs of the VHH antibody are respectively transplanted into the corresponding selected human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CR3-FR4. Preferably, the framework sequence used for replacement has structural similarity with the framework sequence of the antibody to be humanized, for example, having a sequence identity of at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99% or more;

[0170] If necessary, key amino acids in the FR region are back-mutated to amino acids corresponding to the VHH antibody to ensure the original affinity, thereby obtaining a humanized anti-BCMA VHH antibody, and the VHH antibody is optionally sequenced.

[0171] In some embodiments, the back mutation site is selected from one or more of the framework (FR).

[0172] In some embodiments, the heavy chain variable region germline gene suitable for humanization of the VHH antibodies of the invention is selected from IGHV3-53*02 or IGHV3-53*03.

[0173] In some embodiments, the present invention also provides functional variants of single domain antibodies of the present invention (particularly VHH antibodies). The functional variants can be introduced into the encoding nucleic acid sequence of the exemplary single domain antibody of the present invention by methods well known in the present invention, such as by random or site-directed mutagenesis, for example, by introducing CDR sequences and / or FR sequences, and then screening (for example, by phage display library screening) variants that maintain the desired properties to obtain functional variants. Typically, functional variants maintain significant sequence identity with the parent single domain antibody (or VHH). Preferably, the functional variant maintains the desired biological properties of the parent single domain antibody (or VHH), for example, relative to the biological activity of the parent, the variant has a comparable (for example, at least 50%, 60%, 70%, 80%, preferably more than 90%) biological activity, or improved biological activity (for example, 110-150% or higher). The desired biological properties include, for example, but are not limited to, binding affinity to the target antigen (e.g., BCMA) (as measured by a KD value), activity in blocking binding of the target antigen to a receptor (e.g., as measured by an IC50 value), activity in activating T cells in in vitro or in vivo experiments (e.g., as measured by released cytokines), and inhibition of tumor growth / survival in in vitro or in vivo experiments.

[0174] In some embodiments, the present invention provides affinity variants of the VHH polypeptides of the present invention. Preferably, the affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to the parent single domain antibody from which they are derived, wherein the affinity variants have altered binding affinity for the target antigen compared to the parent antibody.

[0175] Heavy chain antibodies

[0176] In another aspect of the present invention, the present invention also provides a heavy chain antibody comprising the heavy chain variable region of the VHH antibody of the present invention.

[0177] In some embodiments, a single domain antibody or VHH of the present invention (e.g., a camel-derived VHH or a humanized form thereof) can be linked to a constant region of a human antibody or a portion thereof, such as an Fc region, to produce a heavy chain antibody comprising a VHH-constant region or VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy chain antibody comprises a VHH antibody of the present invention and an Fc region at its C-terminus. In some embodiments, VHH and Fc are linked by a hinge region or a portion thereof, such as a hinge region from IgG (e.g., a hinge region of IgG1, 2, 3, or 4) or a portion thereof.

[0178] In some embodiments, the anti-BCMA heavy chain antibodies of the present invention comprise a VHH as defined herein, or a heavy chain variable region thereof, and a heavy chain constant region or an Fc region of a heavy chain constant region. In some embodiments, a connecting peptide is included between the VHH or its heavy chain variable region and the heavy chain constant region or Fc region, such as an antibody hinge region or a portion thereof, such as a hinge region or a portion thereof from IgG (including a native or mutated IgG hinge region or a portion thereof).

[0179] In some embodiments, the connecting peptide is a hinge region or portion thereof from human IgG1, 2, 3 or 4, including a native or mutated hinge region or portion thereof, such as a hinge region from human IgG1, for example, the connecting peptide is EPKSS (SEQ ID NO: 94) or EPKSC (SEQ ID NO: 96).

[0180] In one embodiment, the heavy chain antibody comprises an Fc region or portion thereof from a camelid (e.g., an alpaca). In one embodiment, the heavy chain antibody is produced and isolated by immunizing the camelid, e.g., an alpaca. Various methods are known in the art for immunizing camelids and isolating the resulting VHH antibodies or heavy chain antibodies against an antigen of interest.

[0181] In some embodiments, the heavy chain antibody comprises a constant region from a human or non-human primate (eg, cynomolgus monkey) antibody, such as a constant region from human IgG1, human IgG2, human IgG3, or human IgG4.

[0182] In some embodiments, the heavy chain antibody comprises an Fc portion from a human or non-human primate (e.g., cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, e.g., a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region, such as a human IgG1 Fc region.

[0183] In one embodiment, the heavy chain antibody according to the present invention can dimerize with another polypeptide chain (e.g., the same or different heavy chain antibody) comprising an Fc region through the Fc region. Therefore, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising a heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0184] The Fc region of the present invention may be mutated to obtain desired properties. Mutations to the Fc region are known in the art and are defined below.

[0185] In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain comprising a heavy chain variable region, an Fc region, and a connecting peptide connecting the heavy chain variable region and the Fc region. Preferably, the connecting peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 94 or 96.

[0186] In some embodiments, the anti-BCMA antibodies or antigen-binding fragments thereof of the present invention comprise or consist of a heavy chain comprising or consisting of a heavy chain variable region of a VHH of the present invention, a connecting peptide, and an Fc region, wherein the heavy chain

[0187] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 44-46 and 48-55; or

[0188] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 44-46 and 48-55; or

[0189] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 44 to 46 and 48 to 55, preferably, the amino acid changes do not occur in the CDR regions.

[0190] III. Anti-GPRC5D Antibodies

[0191] The present invention also provides a GPRC5D antibody having a higher binding affinity to GPRC5D. In some embodiments, the GPRC5D antibody of the present invention is suitable for constructing an antigen binding region in a multispecific antibody molecule.

[0192] In some embodiments, the anti-GPRC5D antibodies of the invention are single domain antibodies, particularly VHH antibodies.

[0193] Single-domain antibodies, or VHH antibodies, have a molecular weight approximately one-tenth that of a human IgG molecule and a physical diameter of only a few nanometers. Due to their small molecular size, single-domain antibodies offer advantages over conventional four-chain antibodies: high stability and solubility, as well as the ability to recognize hidden antigenic sites. Furthermore, single-domain antibodies are cheaper to prepare than conventional four-chain antibodies. In addition to their use as individual molecules, single-domain antibodies are also suitable components for constructing multispecific molecules.

[0194] Single-domain antibodies, or VHH antibodies, have a molecular weight approximately one-tenth that of a human IgG molecule and a physical diameter of only a few nanometers. Due to their small molecular size, single-domain monoclonal antibodies offer advantages over conventional four-chain antibodies: high stability and solubility, as well as the ability to recognize hidden antigenic sites. Furthermore, single-domain antibodies are cheaper to prepare than conventional four-chain antibodies. In addition to their use as standalone molecules, single-domain antibodies are also suitable components for constructing multispecific molecules.

[0195] In some embodiments, the anti-GPRC5D single-domain antibody of the present invention is a VHH antibody comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region generally has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, and preferably, the boundaries of the three CDRs in the VHH sequence can be defined by IMGT.

[0196] In some embodiments, the anti-GPRC5D VHH antibody of the present invention comprises

[0197] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NOs: 27 or 106, or

[0198] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0199] Preferably, the CDR sequences are defined according to IMGT.

[0200] In some embodiments, the anti-GPRC5D VHH antibody of the present invention comprises or consists of a heavy chain variable region comprising

[0201] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NO: 27 or 106, or

[0202] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0203] Preferably, the CDR sequences are defined according to IMGT.

[0204] In some embodiments, the anti-GPRC5D VHH antibodies of the present invention comprise complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-GPRC5D VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0205] In some embodiments, the VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 24, or the VHH CDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 24.

[0206] In some embodiments, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, or the VHH CDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 25.

[0207] In some embodiments, the VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 26, or the VHH CDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 26.

[0208] In one embodiment, the anti-GPRC5D VHH antibody of the present invention comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 26.

[0209] In one embodiment, an anti-GPRC5D VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein said heavy chain variable region comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26.

[0210] In some embodiments, the anti-GPRC5D VHH antibody of the present invention comprises or consists of a heavy chain variable region, wherein the heavy chain variable region

[0211] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 27 or 106; or

[0212] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 27 or 106; or

[0213] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 27 or 106, preferably, the amino acid changes do not occur in the CDR regions.

[0214] In some embodiments, the anti-GPRC5D VHH antibody of the present invention comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 27 or 106.

[0215] In some embodiments, the VHH antibodies of the present invention comprise CDR amino acid sequences and / or framework (FR) amino acid sequences derived from camelid heavy chain antibodies produced by immunizing camelids (e.g., alpacas). In some embodiments, the VHH monoclonal antibodies of the present invention derived from camelid heavy chain antibodies can be engineered, for example, to comprise framework region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelid mammalian species. In one embodiment, to further improve the properties (e.g., affinity) of the engineered antibodies, camelid amino acid residues located at corresponding positions in the parent camelid antibody can be introduced into the engineered antibodies at one or more positions (e.g., framework regions) by back mutation.

[0216] In one embodiment, the VHH antibody of the invention is a humanized antibody.

[0217] In some embodiments, the present invention also provides functional variants of single domain antibodies of the present invention (particularly VHH antibodies). The functional variants can be introduced into the encoding nucleic acid sequence of the exemplary single domain antibody of the present invention by methods well known in the present invention, such as by random or site-directed mutagenesis, for example, by introducing CDR sequences and / or FR sequences, and then screening (for example, by phage display library screening) variants that maintain the desired properties to obtain functional variants. Typically, functional variants maintain significant sequence identity with the parent single domain antibody (or VHH). Preferably, the functional variant maintains the desired biological properties of the parent single domain antibody (or VHH), for example, relative to the biological activity of the parent, the variant has a comparable (for example, at least 50%, 60%, 70%, 80%, preferably more than 90%) biological activity, or improved biological activity (for example, 110-150% or higher). The desired biological properties include, for example, but are not limited to, binding affinity to the target antigen (e.g., GPRC5D) (e.g., as measured by a KD value), activity in blocking binding of the target antigen to the receptor (e.g., as measured by an IC50 value), activity in activating T cells in in vitro or in vivo experiments (e.g., as measured by released cytokines), and inhibition of tumor growth / survival in in vitro or in vivo experiments.

[0218] In some embodiments, the present invention provides affinity variants of the VHH polypeptides of the present invention. Preferably, the affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to the parent single domain antibody from which they are derived, wherein the affinity variants have altered binding affinity for the target antigen compared to the parent antibody.

[0219] In another aspect of the present invention, the present invention also provides a heavy chain antibody comprising the heavy chain variable region of the anti-GPRC5D VHH antibody of the present invention.

[0220] In some embodiments, the anti-GPRC5D single domain antibody or VHH of the present invention (e.g., camel-derived VHH or a humanized form thereof) can be linked to the constant region of a human antibody or a portion thereof, such as an Fc region, to produce a heavy chain antibody comprising a VHH-constant region or VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy chain antibody comprises a VHH antibody of the present invention and an Fc region at its C-terminus. In some embodiments, the VHH is linked to the Fc region by a hinge region or a portion thereof, such as a hinge region from IgG (e.g., a hinge region of IgG1, 2, 3, or 4) or a portion thereof.

[0221] In some embodiments, the anti-GPRC5D heavy chain antibodies of the present invention comprise an anti-GPRC5D VHH as defined herein, or a heavy chain variable region thereof, and a heavy chain constant region or an Fc region of a heavy chain constant region. In some embodiments, a connecting peptide, such as an antibody hinge region or a portion thereof, such as a hinge region or a portion thereof from IgG (including a native or mutated IgG hinge region or a portion thereof), is included between the anti-GPRC5D VHH or its heavy chain variable region and the heavy chain constant region or Fc region.

[0222] In some embodiments, the connecting peptide is a hinge region or portion thereof from human IgG1, 2, 3 or 4, including a native or mutated hinge region or portion thereof, such as a hinge region from human IgG1, for example, the connecting peptide is EPKSS (SEQ ID NO: 94) or EPKSC (SEQ ID NO: 96).

[0223] In one embodiment, the heavy chain antibody comprises an Fc region or portion thereof from a camelid (e.g., an alpaca). In one embodiment, the heavy chain antibody is produced and isolated by immunizing the camelid, e.g., an alpaca. Various methods are known in the art for immunizing camelids and isolating the resulting VHH antibodies or heavy chain antibodies against an antigen of interest.

[0224] In some embodiments, the heavy chain antibody comprises a constant region from a human or non-human primate (eg, cynomolgus monkey) antibody, such as a constant region from human IgG1, human IgG2, human IgG3, or human IgG4.

[0225] In some embodiments, the heavy chain antibody comprises an Fc portion from a human or non-human primate (e.g., cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, e.g., a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region, such as a human IgG1 Fc region.

[0226] In one embodiment, the heavy chain antibody according to the present invention can dimerize with another polypeptide chain (e.g., the same or different heavy chain antibody) comprising an Fc region through the Fc region. Therefore, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising a heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0227] The Fc region of the present invention may be mutated to obtain desired properties. Mutations to the Fc region are known in the art and are defined below.

[0228] In some embodiments, the anti-GPRC5D antibody of the present invention comprises a heavy chain comprising a heavy chain variable region, an Fc region, and a connecting peptide connecting the heavy chain variable region and the Fc region. Preferably, the connecting peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 94 or 96.

[0229] V. Multispecific Antibodies

[0230] In some embodiments, the anti-BCMA antibodies of the invention are multispecific antibodies, e.g., bispecific antibodies or trispecific antibodies, e.g., comprising one binding specificity for BCMA and other binding specificities for one or more other molecules, e.g., CD3, or e.g., CD3 and GPRC5D.

[0231] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0232] a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to CD3, and / or the second antigen-binding region specifically binds to BCMA.

[0233] Therefore, another aspect of the present invention relates to a trispecific antibody comprising

[0234] A first antigen-binding region, a second antigen-binding region, and a third antigen-binding region, wherein the first antigen-binding region specifically binds CD3, the second antigen-binding region specifically binds BCMA, and the third antigen-binding region specifically binds GPRC5D.

[0235] In some embodiments, the first antigen-binding region is from an anti-CD3 antibody, such as the SP34 antibody or a humanized antibody thereof, such as the anti-CD3 humanized antibody disclosed in PCT / CN2023 / 071314, such as the Fab fragment of the anti-CD3 antibody or the scFv fragment of the anti-CD3 antibody.

[0236] In some embodiments, the second antigen-binding region is from an anti-BCMA antibody described herein, eg, an anti-BCMA VHH.

[0237] In some embodiments, the third antigen-binding region is from an anti-GPRC5D antibody, such as an anti-GPRC5D antibody disclosed in PCT / CN2023 / 071314, for example, a Fab fragment of an anti-GPRC5D antibody. In some embodiments, the third antigen-binding region is from an anti-GPRC5D VHH antibody, such as a VHH antibody described above.

[0238] The first antigen-binding region of the multispecific antibody suitable for use in the present invention may comprise or consist of a full-length anti-CD3 antibody or an antigen-binding fragment thereof (e.g., the humanized anti-CD3 antibody or an antigen-binding fragment thereof disclosed in PCT / CN2023 / 071314), as long as it can specifically bind to CD3, including but not limited to, full-length antibodies, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH, or heavy chain antibodies that specifically bind to CD3.

[0239] The second antigen-binding region of the multispecific antibody suitable for the present invention may comprise or consist of an anti-BCMA VHH of the present invention, as long as it can specifically bind to BCMA, including but not limited to, for example, a full-length antibody, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy chain antibody that specifically binds to BCMA.

[0240] The third antigen-binding region of the multispecific antibody suitable for use in the present invention may comprise, for example, an anti-GPRC5D VHH of the present invention, or be composed thereof, or comprise, or be composed of, an anti-GPRC5D full-length antibody or an antigen-binding fragment thereof (e.g., an anti-GPRC5D antibody or an antigen-binding fragment thereof disclosed in PCT / CN2023 / 071314), as long as it can specifically bind to GPRC5D, including but not limited to, for example, a full-length antibody, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy chain antibody that specifically binds to GPRC5D.

[0241] In some embodiments, the bispecific antibodies or trispecific antibodies of the present invention are IgG-like bispecific antibodies. "IgG-like bispecific antibodies" as described herein refer to bispecific antibodies or trispecific antibodies that comprise an Fc dimer. Therefore, in some embodiments, the bispecific antibodies or trispecific antibodies of the present invention comprise an Fc dimer.

[0242] In some embodiments, the bispecific antibodies of the present invention are IgG-like bispecific antibodies, which comprise an scFv fragment as an antigen-binding region that specifically binds to one antigen and a VHH as an antigen-binding region that specifically binds to another antigen. In some embodiments, the IgG-like bispecific antibody comprises an scFv that specifically binds to CD3 as a first antigen-binding region and a VHH fragment that specifically binds to BCMA as a second antigen-binding region.

[0243] In one embodiment, the bispecific antibody may comprise one or more first antigen-binding regions. In one embodiment, the bispecific antibody may comprise one or more second antigen-binding regions. In one embodiment, the bispecific antibody comprises a first antigen-binding region and one or two (e.g., in series) second antigen-binding regions.

[0244] In some embodiments, the trispecific antibodies of the present invention are IgG-like trispecific antibodies, which comprise a Fab fragment as an antigen-binding region that specifically binds to one antigen, and a VHH as an antigen-binding region that specifically binds to two other antigens. In some embodiments, the IgG-like trispecific antibody comprises a first antigen-binding region comprising or consisting of a Fab fragment that specifically binds to CD3, a second antigen-binding region comprising or consisting of a VHH fragment that specifically binds to BCMA, and a third antigen-binding region comprising or consisting of a VHH fragment that specifically binds to GPRC5D.

[0245] In some embodiments, the trispecific antibodies of the present invention are IgG-like trispecific antibodies, which comprise an scFv fragment as an antigen-binding region that specifically binds to one antigen, a VHH fragment as an antigen-binding region that specifically binds to another antigen, and a Fab fragment as an antigen-binding region that specifically binds to a third antigen. In some embodiments, the IgG-like trispecific antibody comprises a first antigen-binding region comprising or consisting of an scFv fragment that specifically binds to CD3, a second antigen-binding region comprising or consisting of a VHH fragment that specifically binds to BCMA, and a third antigen-binding region comprising or consisting of a Fab fragment that specifically binds to GPRC5D.

[0246] In one embodiment, the trispecific antibody may comprise one or more first antigen-binding regions. In one embodiment, the trispecific antibody may comprise one or more second antigen-binding regions. In one embodiment, the trispecific antibody may comprise one or more third antigen-binding regions. In one embodiment, the trispecific antibody comprises a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region.

[0247] In some embodiments, the first antigen-binding region that specifically binds to CD3 is selected from scFv or Fab, the second antigen-binding region that specifically binds to BCMA is VHH, and / or the third antigen-binding region that specifically binds to GPRC5D is selected from VHH or Fab.

[0248] Fab fragments of the multispecific antibodies suitable for use in the present invention

[0249] In some embodiments, the Fab fragment, which is one of the antigen-binding regions of a multispecific antibody, is composed of two polypeptide chains comprising the antibody VH, CH1 (optionally further comprising a hinge region), VL, and CL domains, wherein the VH is paired with the VL and the CH1 is paired with the CL to form an antigen-binding region. In some embodiments, in the Fab, one chain comprises VH and CH1 from N-terminus to C-terminus (i.e., VH-CH1), and the other chain comprises VL and CL from N-terminus to C-terminus (i.e., VL-CL).

[0250] In some embodiments, in the multispecific antibody, Fab can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising VH, wherein the Fc domain may or may not include a hinge region (e.g., EPKSS or EPKSC). Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and is fused to the antibody Fc domain via the C-terminus of CH1 of the VH-CH1 chain, wherein the Fc domain may or may not include a hinge region (e.g., EPKSS or EPKSC). Herein, the Fab chain connected to the Fc dimer is also referred to as the Fab heavy chain, and the Fab chain not connected to the Fc dimer is also referred to as the Fab light chain. In some embodiments, the fusion is direct fusion or fusion via a linker.

[0251] In some embodiments, the CH1 of a Fab may further comprise a partial hinge region, such as EPKSS or EPKSC, to facilitate formation of a stable structure, for example, to facilitate the preparation of multispecific antibodies. When the Fc fused to the Fab heavy chain does not comprise this partial hinge region, or when the Fab heavy chain is fused to a non-Fc domain, the C-terminus of the CH1 may comprise this partial hinge region to facilitate formation of a stable structure. When the Fc fused to the Fab heavy chain comprises this partial hinge region, the C-terminus of the CH1 of the Fab heavy chain may not comprise this partial hinge region.

[0252] In some embodiments, the Fab comprised in the multispecific antibody of the invention specifically binds CD3 or specifically binds GPRC5D.

[0253] In some embodiments, the Fab heavy chain comprises VH and CH1 (and optionally comprises part of the hinge region EPKSS or EPKSC at the C-terminus of CH1). In some embodiments, the CH1 is a CH1 from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 from IgG1. In some embodiments, the CH1

[0254] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 88;

[0255] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 88; or

[0256] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 88.

[0257] In some embodiments, the Fab light chain comprises VL-CL.In some embodiments, the CL is a light chain constant region from an antibody kappa or lambda light chain, preferably a light chain constant region from a kappa light chain.

[0258] scFv suitable for the multispecific antibodies of the present invention

[0259] In some embodiments, the scFv fragment as one of the multispecific antibody binding regions is composed of a polypeptide chain comprising the VH and VL domains of the antibody, wherein the VH and VL are connected (e.g., via a linker) to pair to form an antigen binding site. In some embodiments, the scFv is in a trans configuration and comprises, from N-terminus to C-terminus: VH, a linker, and VL (VH-linker-VL). In other embodiments, the scFv is in a cis configuration and comprises, from N-terminus to C-terminus: VL, a linker, and VH (VL-linker-VH). In some embodiments, the linker is a peptide linker consisting of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acids in length, e.g., 5-30 amino acids in length, e.g., 15 amino acids or 20 amino acids in length. In one embodiment, the linker comprises the amino acid sequence (G4S)n, wherein n=1, 2, 3, 4 or 5, preferably, n=3 or 4, more preferably, n=3.

[0260] In some preferred embodiments, the scFv antigen binding site contained in the antibody molecule of the invention is a disulfide-stabilized scFv.

[0261] In some embodiments, in the multispecific antibodies, the scFv can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain. In some embodiments, in the multispecific antibodies, the scFv is fused to the C-terminus of another antigen binding region (e.g., a Fab heavy chain) at the N-terminus of the chain and to the N-terminus of the Fc domain of the antibody at the C-terminus of the chain.

[0262] In some embodiments, in the multispecific antibodies, the scFv can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising VL. In some embodiments, in the multispecific antibodies, the scFv is fused to the C-terminus of another antigen binding region (e.g., a Fab heavy chain) at the N-terminus of the VH chain and to the N-terminus of the Fc domain of the antibody at the C-terminus of the VL chain.

[0263] In some embodiments, in the multispecific antibodies, the scFv can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising VH. In some embodiments, in the multispecific antibodies, the scFv is fused to the C-terminus of another antigen binding region (e.g., a Fab heavy chain) at the N-terminus of the VL chain and to the N-terminus of the Fc domain of the antibody at the C-terminus of the VH chain.

[0264] In some embodiments, the scFv comprised in the multispecific antibodies of the invention specifically binds CD3.

[0265] Fc dimers suitable for the multispecific antibodies of the present invention

[0266] In one embodiment, the two Fc regions in the multispecific antibody of the present invention dimerize to form a dimeric Fc. Preferably, the two Fc regions heterodimerize to form a heterodimeric Fc.

[0267] In some embodiments, the first and second Fc regions are identical. In other embodiments, the first and second Fc regions are different and pair and heterodimerize.

[0268] The Fc region fragment suitable for antibody molecules of the present invention can be any antibody Fc region. The Fc region can include native sequence Fc regions and variant Fc regions. The native sequence Fc domain covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc regions of their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of an antibody of the present invention can include two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. In some embodiments, the antibody Fc region can also have an IgG hinge region or a partial IgG hinge region at the N-terminus, such as an IgG1 hinge region or a partial IgG1 hinge region. Mutations can be contained in the hinge region. In some embodiments, the partial hinge region can be EPKSS or EPKSC.

[0269] Preferably, the Fc region of an antibody of the present invention comprises, from N-terminus to C-terminus, CH2-CH3, or, from N-terminus to C-terminus, hinge region-CH2-CH3. In some embodiments, the Fc region suitable for use in an antibody or multispecific antibody of the present invention is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3, or human IgG4 Fc. In one embodiment, the Fc region is derived from a human IgG1 Fc, e.g., comprising or consisting of an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or greater identity thereto.

[0270] The Fc region of the antibody or multispecific antibody of the present invention can be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0271] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0272] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector function, eg, comprises a mutation that achieves the above function.

[0273] As will be appreciated by those skilled in the art, according to the intended use of the antibody molecule of the present invention, the antibody molecule of the present invention may also include modifications in the Fc domain that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used for the present invention has one or more of the L234A / L235A mutation, the D265A mutation, or the P329A mutation that reduces binding to the Fcγ receptor. In some embodiments, the Fc region used for the present invention has a L234A / L235A mutation, the D265A mutation, and the P329A mutation that reduces binding to the Fcγ receptor. In another preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn. In some embodiments, the Fc region comprising a mutation that reduces binding to an Fcγ receptor comprises or consists of the amino acid sequence of SEQ ID NO: 90 or 97, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 90 or 97 and comprises an L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0274] As will be appreciated by those skilled in the art, to promote the formation of heterodimers of the multispecific antibodies of the present invention, the Fc regions comprised by the multispecific antibodies of the present invention may comprise mutations that are conducive to heterodimerization. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions.

[0275] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are engineered in a complementary manner such that each CH3 region (or a heavy chain comprising it) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementary engineered CH3 region (such that the first and second CH3 regions heterodimerize and homodimers are not formed between the two first CH3 regions or the two second CH3 regions).

[0276] Preferably, corresponding knob mutations and hole mutations are introduced into the first monomer Fc region and the second monomer Fc region, respectively, based on the Knob-in-Hole technique. For this technique, see, for example, Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.

[0277] In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index).

[0278] In a further embodiment, in the CH3 region of one Fc region, the knob mutation comprises or consists of substitution of the threonine residue at position 366 with a tryptophan residue (T366W) and substitution of the serine residue at position 354 with a cysteine ​​residue (S354C) or substitution of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (in particular, substitution of the serine residue at position 354 with a cysteine ​​residue); and in the CH3 region of another Fc region, the hole mutation comprises or consists of substitution of the tyrosine residue at position 407 with a valine residue (Y407V), optionally substitution of the threonine residue at position 366 with a serine residue (T366S) and substitution of the leucine residue at position 368 with an alanine residue (L368A) (numbering according to the EU index), optionally substitution of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0279] In a specific embodiment, one Fc region comprises amino acid substitutions S354C and T366W (knob mutation), and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (hole mutation) (numbering is according to the EU index).

[0280] Thus, in a specific embodiment, the multispecific antibody of the present invention comprises two Fc regions that heterodimerize, wherein

[0281] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0282] b) one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0283] c) one Fc-region polypeptide comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C;

[0284] Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, eg, one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; eg, a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0285] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0286] In a specific embodiment, the two Fc regions of the multispecific antibody of the invention heterodimerize, wherein

[0287] The first Fc region comprises a knob mutation comprising, or consisting of, an amino acid sequence of SEQ ID NO: 47, 86, or 87, or an amino acid sequence at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical thereto; in some embodiments, the Fc region comprises an amino acid sequence at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 47, 86, or 87 and comprises a knob mutation (e.g., S354C and T366W); in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC;

[0288] The second Fc region comprises a hole mutation, which comprises or consists of an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or higher identity, to an amino acid sequence of SEQ ID NO: 84, 85 or 111. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or higher identity, to SEQ ID NO: 84, 85 or 111 and comprises a hole mutation (e.g., T366S, L368A, Y407V, and Y349C); in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC.

[0289] Antigen binding region that specifically binds to CD3

[0290] In some embodiments, the multispecific antibodies of the present invention comprising an antigen-binding region that specifically binds CD3 bind to CD3 (e.g., human CD3 or monkey CD3, such as cynomolgus CD3) with a desired affinity. In some embodiments, the multispecific antibodies of the present invention comprising an antigen-binding region that specifically binds CD3 bind to both human CD3 and monkey CD3, such as cynomolgus CD3. In some embodiments, the affinity of the antibodies is determined by thin-layer interferometry or surface plasmon resonance.

[0291] In some embodiments, the multispecific antibodies of the present invention comprising an antigen-binding region that specifically binds to CD3 have an equilibrium dissociation constant (K D ) binds to human CD3 or monkey CD3, such as cynomolgus monkey CD3, said K D In some embodiments, the multispecific antibodies of the present invention comprising an antigen-binding region that specifically binds to CD3 have a K of about 10 nM to 100 nM, or 20 nM to 100 nM, or 50 nM to 100 nM. D Binds to monkey CD3, such as cynomolgus monkey CD3. In some embodiments, the multispecific antibody of the invention comprising an antigen binding region that specifically binds to CD3 has a K between about 100-1000 nM (e.g., between about 200, 300, 400, or 500-1000 nM). D Binds to human CD3.

[0292] In some embodiments, the multispecific antibodies of the present invention comprising an antigen binding region that specifically binds CD3 bind to CD3 on the surface of effector cells. In some embodiments, the multispecific antibodies of the present invention comprising an antigen binding region that specifically binds CD3 can activate effector cells. In some embodiments, the effector cells are T cells. In some embodiments, the binding is detected using flow cytometry. In some embodiments, the activation effect of the antibody is detected using a reporter gene detection system (e.g., the Jurkat / NFAT-luc reporter gene system).

[0293] In some embodiments, the multispecific antibodies of the present invention comprising an antigen binding region that specifically binds to CD3 can activate effector cells to induce killing of tumor cells.

[0294] In some embodiments, the antigen binding region of the present invention that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0295] In some embodiments, the antigen binding region of the present invention that specifically binds to CD3 comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0296] In some embodiments, the antigen binding region of the present invention that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0297] In some aspects, the antigen binding region that specifically binds to CD3 of the present invention comprises a heavy chain variable region (VH). In some aspects, the antigen binding region that specifically binds to CD3 of the present invention comprises a light chain variable region (VH). In some aspects, the antigen binding region that specifically binds to CD3 of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0298] In some embodiments, the antigen-binding region that specifically binds to CD3 of the present invention further comprises an antibody heavy chain constant region HC. In some embodiments, the antigen-binding region that specifically binds to CD3 of the present invention further comprises an antibody light chain constant region LC. In some embodiments, the antigen-binding region that specifically binds to CD3 of the present invention further comprises a heavy chain constant region HC and a light chain constant region LC.

[0299] In some embodiments, the heavy chain variable region VH of the antigen binding region that specifically binds to CD3 of the present invention is

[0300] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 34; or

[0301] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 34; or

[0302] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 34, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0303] In some embodiments, the antigen-binding region light chain variable region VL of the present invention that specifically binds to CD3

[0304] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 35; or

[0305] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 35; or

[0306] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 35, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0307] In some embodiments, the three complementarity determining regions (HCDRs) of the heavy chain variable region of the antigen binding region that specifically binds to CD3 of the present invention, HCDR1, HCDR2 and HCDR3 are

[0308] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 34, or

[0309] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of (i),

[0310] Preferably, the HCDRs are defined according to Kabat.

[0311] In some embodiments, the three complementarity determining regions (LCDRs) of the light chain variable region of the antigen binding region that specifically binds to CD3, LCDR1, LCDR2, and LCDR3, are

[0312] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 35, or

[0313] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of (i);

[0314] Preferably, the LCDR is determined according to Kabat.

[0315] In some embodiments, in the antigen binding region of the present invention that specifically binds to CD3,

[0316] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 28; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 29; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 30; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 31; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 32; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 33.

[0317] In some specific embodiments of the present invention, the antigen binding region that specifically binds to CD3 of the present invention comprises VH and VL, wherein

[0318] The VH comprises the amino acid sequence of SEQ ID NO:34, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO:35, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0319] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 of the present invention comprises the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:34, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:35.

[0320] In some specific embodiments of the present invention, the antigen binding region that specifically binds to CD3 of the present invention comprises: HCDR1 as shown in SEQ ID NO:28, HCDR2 as shown in SEQ ID NO:29, HCDR3 as shown in SEQ ID NO:30; LCDR1 as shown in SEQ ID NO:31, LCDR2 as shown in SEQ ID NO:32 and LCDR3 as shown in SEQ ID NO:33.

[0321] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0322] In some embodiments, the antigen-binding region of the present invention that specifically binds to CD3 comprises a heavy chain constant region or fragment thereof (e.g., CH1) that is an IgG1, IgG2, IgG3, or IgG4 constant region or fragment thereof. In some embodiments, the antigen-binding region of the present invention that specifically binds to CD3 comprises a light chain constant region or fragment thereof that is a kappa or lambda light chain constant region or fragment thereof, such as a lambda light chain constant region or fragment thereof.

[0323] In one embodiment, the antigen-binding region that specifically binds to CD3 of the present invention is a fragment of an anti-CD3 antibody (e.g., an antigen-binding fragment), preferably an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody.

[0324] In one embodiment, the antigen binding region that specifically binds to CD3 of the present invention is a Fab, which comprises the heavy chain variable region VH and the light chain variable region VL described in this section.

[0325] In some embodiments, the Fab heavy chain comprises VH and CH1 (and optionally part of the hinge region EPKSS or EPKSC at the C-terminus of CH1), wherein VH is the VH of an anti-CD3 antibody.

[0326] In some embodiments, the Fab light chain comprises VL-CL, wherein VL is the VL of an anti-CD3 antibody. In some embodiments, the CL is a light chain constant region from an antibody kappa or lambda light chain, preferably a light chain constant region from a kappa light chain.

[0327] In some embodiments, the Fab heavy chain

[0328] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 11;

[0329] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 11; or

[0330] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 11.

[0331] In some embodiments, the Fab light chain

[0332] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 60;

[0333] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 60; or

[0334] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 60.

[0335] In a specific embodiment, the antigen binding region of the present invention that specifically binds to CD3 is a scFv, which comprises the heavy chain variable region VH and light chain variable region VL as described above. In some embodiments, the scFv comprises the heavy chain variable region VH described in this section, a linker, and the light chain variable region described in this section from the N-terminus to the C-terminus. In some embodiments, the scFv comprises the sequence as shown in SEQ ID NO: 109 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 109 and having the same CDRs.

[0336] Antigen binding region that specifically binds to GPRC5D

[0337] The antigen binding region that specifically binds to GPRC5D is derived from an anti-GPRC5D single domain antibody

[0338] In some embodiments, the antigen binding region of the antibody is derived from a VHH antibody that specifically binds GPRC5D, such as an anti-GPRC5D VHH as described herein.

[0339] The antigen binding region is derived from an anti-GPRC5D full-length antibody or an antigen-binding fragment thereof

[0340] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to GPRC5D, such as an antibody or antigen-binding fragment thereof that specifically binds to GPRC5D disclosed in PCT / CN2023 / 071314.

[0341] In some embodiments, the antibody that specifically binds to GPRC5D comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO:42.

[0342] In some embodiments, the antibody that specifically binds to GPRC5D comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are the LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO:43.

[0343] In some embodiments, the HCDR1 of the antibody that specifically binds to GPRC5D comprises or consists of the amino acid sequence of SEQ ID NO:36; the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:37, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:38, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:39, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:40, and / or the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:41.

[0344] In some embodiments, an antibody that specifically binds to GPRC5D comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:36; HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:37, HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:38, LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:39, LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:40, and LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:41.

[0345] In some aspects, an antibody that specifically binds to GPRC5D comprises a heavy chain variable region (VH), wherein the VH

[0346] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 42;

[0347] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 42; or

[0348] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 42, preferably, the amino acid changes do not occur in the CDR regions.

[0349] In some aspects, an antibody that specifically binds to GPRC5D comprises a light chain variable region (VL), wherein the VL

[0350] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 43;

[0351] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 43; or

[0352] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 43, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0353] In some embodiments, the antibody that specifically binds to GPRC5D comprises a VH and a VL, wherein the VH comprises or consists of the sequence shown in SEQ ID NO: 42; and / or the VL comprises or consists of the sequence shown in SEQ ID NO: 43.

[0354] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody (eg, an antibody that specifically binds to GPRC5D disclosed in PCT / CN2023 / 071314) or an antibody that specifically binds to GPRC5D described above.

[0355] In some embodiments, the antigen binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of known (e.g., antibodies that specifically bind to GPRC5D disclosed in PCT / CN2023 / 071314) or the above-mentioned antibodies that specifically bind to GPRC5D.

[0356] In some embodiments, the antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of known (e.g., antibodies that specifically bind to GPRC5D disclosed in PCT / CN2023 / 071314) or the above-mentioned antibodies that specifically bind to GPRC5D.

[0357] In some embodiments, the antigen binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody (eg, an antibody that specifically binds to GPRC5D disclosed in PCT / CN2023 / 071314) or the above-mentioned antibody that specifically binds to GPRC5D.

[0358] In some embodiments, the antigen binding region comprises the heavy chain variable region and light chain variable region of a known antibody (e.g., an antibody that specifically binds to GPRC5D disclosed in PCT / CN2023 / 071314) or the above-mentioned antibody that specifically binds to GPRC5D, and the mutations described herein.

[0359] Some antigen-binding regions comprise Fab of a known antibody (eg, an antibody specifically binding to GPRC5D disclosed in PCT / CN2023 / 071314) or the above-mentioned antibody specifically binding to GPRC5D.

[0360] In one embodiment, the antigen binding region that specifically binds to GPRC5D of the present invention is a Fab comprising the heavy chain variable region VH and the light chain variable region VL described in this section.

[0361] In some embodiments, the Fab heavy chain comprises VH and CH1 (and optionally part of the hinge region EPKSS or EPKSC at the C-terminus of CH1), wherein VH is the VH of an anti-GPRC5D antibody.

[0362] In some embodiments, the Fab light chain comprises VL-CL, wherein VL is the VL of an anti-GPRC5D antibody. In some embodiments, the CL is a light chain constant region from an antibody kappa or lambda light chain, preferably a light chain constant region from a kappa light chain.

[0363] In some embodiments, the Fab heavy chain

[0364] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 110;

[0365] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 110; or

[0366] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 110.

[0367] In some embodiments, the Fab light chain

[0368] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 62;

[0369] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 62; or

[0370] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 62.

[0371] Exemplary bispecific antibody molecules

[0372] In some preferred embodiments, the present invention provides a bispecific antibody comprising a VHH fragment that specifically binds to BCMA and an antigen binding region that specifically binds to CD3, and optionally an Fc region.

[0373] In some embodiments, the present invention provides a bispecific antibody comprising a first antigen-binding region, a second antigen-binding region, and an Fc dimer, wherein the first antigen-binding region is a Fab fragment or scFv fragment that specifically binds to CD3, and the second antigen-binding region is a VHH that specifically binds to BCMA.

[0374] Therefore, in some embodiments, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody, comprising one or two anti-BCMA VHHs, an anti-CD3 antibody scFv and an Fc heterodimer, wherein

[0375] The scFv comprises VH-VL (optionally, the VH and VL are fused via a linker), the VHH comprises or consists of a heavy chain variable region,

[0376] wherein the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the first Fc region (eg, comprising a knob mutation or a hole mutation) to constitute the first heavy chain;

[0377] The C-terminus of the one anti-BCMA VHH or two tandem anti-BCMA VHHs is fused to a second Fc region (eg, comprising a hole mutation or a knob mutation) to form a second heavy chain (eg, the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region).

[0378] In some specific embodiments, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising one or two anti-BCMA VHHs, an anti-CD3 antibody scFv and an Fc heterodimer, wherein

[0379] The scFv comprises VH-VL (optionally, the VH and VL are fused via a linker), the VHH comprises or consists of a heavy chain variable region,

[0380] wherein the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the first Fc region comprising a knob mutation, constituting the first heavy chain;

[0381] The C-terminus of the one anti-BCMA VHH or two tandem anti-BCMA VHHs is fused to a second Fc region comprising a hole mutation to constitute a second heavy chain, wherein, for example, the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region).

[0382] In some embodiments, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody, comprising an anti-BCMA VHH or two tandem anti-BCMA VHHs, an anti-CD3 antibody Fab and an Fc heterodimer, wherein

[0383] The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region,

[0384] wherein the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the first Fc region (eg, comprising a knob mutation or a hole mutation) to constitute the first heavy chain;

[0385] The C-terminus of the one anti-BCMA VHH or two tandem anti-BCMA VHHs is fused to a second Fc region (eg, comprising a hole mutation or a knob mutation) to form a second heavy chain (eg, the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region).

[0386] In some embodiments, the Fc region may or may not include a hinge region. In some embodiments, the hinge region is selected from EPKSS or EPKSC. In some embodiments, the fusion is direct fusion or fusion via a linker or connecting peptide. In some embodiments, the connecting peptide is selected from (GGGGS)n, where n=1, 2, 3, or 4.

[0387] In some embodiments, the first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0388] The second heavy chain comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 56, 102, 103 or 104, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0389] In some embodiments, the bispecific antibody comprises or consists of a first heavy chain and a second heavy chain described herein.

[0390] Exemplary trispecific antibodies

[0391] In some embodiments, the present invention provides a trispecific antibody comprising a first antigen-binding region that specifically binds CD3, a second antigen-binding region that specifically binds BCMA, and a third antigen-binding region that specifically binds GPRC5D, and an Fc dimer, such as an Fc heterodimer.

[0392] In some embodiments, the first antigen-binding region that specifically binds to CD3 comprises or consists of a Fab fragment or a scFv fragment of an anti-CD3 antibody.

[0393] In some embodiments, the second antigen-binding region that specifically binds BCMA comprises or consists of an anti-BCMA VHH antibody.

[0394] In some embodiments, the third antigen binding region that specifically binds to GPRC5D comprises or consists of a GPRC5D VHH antibody, or comprises or consists of an anti-GPRC5D Fab fragment.

[0395] In some embodiments, the present invention provides a trispecific antibody comprising an anti-GPRC5D VHH, an anti-BCMA VHH, an anti-CD3 Fab fragment, and an Fc dimer, wherein

[0396] The Fab fragment comprises VH-CH1 (optionally containing part of the hinge region EPKSS or EPKSC) and VL-CL, wherein the VHH comprises a heavy chain variable region, and the trispecific antibody comprises a first heavy chain, a second heavy chain and a light chain, or consists of said chains, wherein

[0397] The first heavy chain comprises, from N-terminus to C-terminus: an anti-GPRC5D VHH, an anti-BCMA VHH, and a first Fc region (e.g., comprising a hole or knob mutation) fused via a linker or not; or an anti-BCMA VHH, an anti-GPRC5D VHH, and a first Fc region (e.g., comprising a hole or knob mutation) fused via a linker or not;

[0398] The second heavy chain comprises, from N-terminus to C-terminus, an anti-CD3 Fab fragment heavy chain and a second Fc region (e.g., comprising a knob or hole mutation) fused via or without a linker;

[0399] Light chain: anti-CD3 Fab fragment light chain;

[0400] For example, the structure is shown in FIG7A .

[0401] In some embodiments, the trispecific antibody comprises or consists of a first heavy chain, a second heavy chain and a light chain, wherein

[0402] the first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;

[0403] The second heavy chain comprises, or consists of, the amino acid sequence of any one of SEQ ID NO: 59, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and / or

[0404] The light chain comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:60, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0405] In some embodiments, the present invention provides a trispecific antibody comprising an anti-GPRC5D Fab fragment, an anti-BCMA VHH, an anti-CD3 scFv, and an Fc dimer, wherein

[0406] The Fab fragment comprises VH-CH1 (optionally containing part of the hinge region EPKSS or EPKSC) and VL-CL, the VHH comprises a heavy chain variable region, the scFV comprises VH-VL, and the trispecific antibody comprises a first heavy chain, a second heavy chain and a light chain, or consists of said chains, wherein

[0407] The first heavy chain comprises, from N-terminus to C-terminus, an anti-BCMA VHH and a first Fc region (e.g., comprising a hole or knob mutation) fused with or without a linker;

[0408] The second heavy chain comprises, from N-terminus to C-terminus, an anti-GPRC5D Fab fragment heavy chain, an anti-CD3 scFv, and a second Fc region (e.g., comprising a knob or hole mutation) fused via a linker or not;

[0409] Light chain: light chain of Fab fragment against GPRC5D;

[0410] For example, the structure is shown in FIG7B .

[0411] In some embodiments, the trispecific antibody comprises or consists of a first heavy chain, a second heavy chain and a light chain, wherein

[0412] The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:56, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;

[0413] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0414] The light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0415] In some embodiments, the Fc region may or may not include a hinge region. In some embodiments, the hinge region is selected from EPKSS or EPKSC. In some embodiments, the fusion is direct or via a linker or connecting peptide. In the case where CH1 comprises a portion of the hinge region EPKSS or EPKSC and is connected to the Fc region, the Fc region need not include that portion of the hinge region.

[0416] In some embodiments, the connecting peptide is selected from (GGGGS)n or (GGSGG)n, wherein n=1, 2, 3 or 4.

[0417] In some embodiments, the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation. In some embodiments, the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation.

[0418] In some embodiments, one or both of the first and second Fc regions comprise L234A and L235A mutations and / or D265A and / or P329A. In some embodiments, the first Fc region comprises a knob mutation and L234A and L235A mutations and D265A and P329A, and the second Fc region comprises a hole mutation and L234A and L235A mutations and D265A and P329A. In some embodiments, the first Fc region comprises a hole mutation and L234A and L235A mutations and D265A and P329A, and the second Fc region comprises a knob mutation and L234A and L235A mutations and D265A and P329A.

[0419] VI. Nucleic acids encoding antibodies and host cells containing the same

[0420] In one aspect, the invention provides nucleic acids encoding any of the above anti-BCMA antibodies or multispecific antibodies, or any one chain thereof.

[0421] For example, nucleic acids of the invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22, 23, 44-46, 48-55, 56, 58, 59, 61, 102-104, or nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22, 23, 44-46, 48-55, 56, 58, 59, 61, 102-104 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0422] As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding molecules of the present invention can be produced by methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0423] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or any antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human BCMA antigen binding ability.

[0424] In yet another aspect, the present invention provides nucleic acids encoding multispecific antibodies. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of displaying binding ability to human or monkey (e.g., cynomolgus monkey) CD3 and human BCMA antigens, and optionally human or monkey (e.g., cynomolgus monkey) GPRC5D antigens and. In one embodiment, the nucleic acids encoding each chain of the multispecific antibody may be in the same vector or in different vectors. In yet another embodiment, the nucleic acids encoding each chain of the multispecific antibody may be introduced into the same or different host cells for expression. Therefore, in some embodiments, the method for producing the multispecific antibody of the present invention comprises the steps of culturing a host cell containing nucleic acids encoding each chain under conditions suitable for expressing each chain of the molecule to produce the multispecific antibody of the present invention.

[0425] In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0426] In one embodiment, a host cell comprising the nucleic acid or the vector is provided, for example, a vector for cloning or expressing an anti-BCMA antibody or a multispecific antibody. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells such as CHO cells (e.g., CHO-S, such as ExpiCHO-S) or 293 cells (e.g., 293F or HEK293 cells) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0427] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F, or 293T cells); and the like. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, and the like; and myeloma cell lines such as Y0, NS0, and Sp2 / 0. Suitable mammalian host cell lines for producing antibodies are known in the art.

[0428] VII. Production and Purification of Anti-BCMA Antibodies or Multispecific Antibodies of the Invention

[0429] In one embodiment, a method of preparing an anti-BCMA antibody or multispecific antibody of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-BCMA antibody or multispecific antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for expression of the anti-BCMA antibody or multispecific antibody or its chains, and optionally recovering the anti-BCMA antibody or multispecific antibody from the host cell (or host cell culture medium).

[0430] The polynucleotide encoding the polypeptide chains of the anti-BCMA antibodies or multispecific antibodies of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0431] The anti-BCMA antibodies or multispecific antibodies prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0432] The purity of the antibody molecules of the present invention can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0433] VIII. Assays for Anti-BCMA Antibodies or Multispecific Antibodies

[0434] The anti-BCMA antibodies or multispecific antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0435] In one aspect, the anti-BCMA antibodies or multispecific antibodies of the present invention are tested for their target (e.g., antigen, e.g., free antigen or antigen expressed on cells) binding activity, for example, by known methods such as thin-layer interferometry, ELISA, flow cytometry, etc. Binding to CD3 and / or BCMA and / or GPRC5D (or CD3 and / or BCMA and / or GPRC5D expressed on cells) can be determined using methods known in the art, exemplary methods are disclosed herein. In some embodiments, radioimmunoassay (RIA) or thin-layer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS) is used for measurement.

[0436] The present invention also provides an assay for identifying the biological activity of an anti-BCMA antibody or multispecific antibody. The biological activity is selected from the properties of the anti-BCMA antibody or multispecific antibody of the present invention.

[0437] For example, the binding activity of the antibody molecules of the present invention to cells expressing BCMA and / or GPRC5D can be determined by methods known in the art, such as fluorescent reporter molecules and flow cytometry, or the exemplary methods disclosed in the Examples herein, such as determining the binding of the antibody molecules of the present invention to BCMA and / or GPRC5D expressed on cells.

[0438] For example, the T cell activation activity of the antibody molecules of the present invention can be determined by methods known in the art, such as a T cell activation test system, such as a NFAT-luc reporter gene system such as a Jurkat / NFAT-luc reporter gene system, for example, by detecting the CD3 signaling pathway in T cells or by detecting the release of cytokines (e.g., interferons, such as IFNγ; tumor necrosis factors, such as TNFα and / or interleukins, such as IL-6) after activation of T cells by the methods shown in the Examples.

[0439] For example, the tumor inhibition activity or structural safety of the antibody molecules of the present invention can be determined by methods known in the art, such as tumor inhibition experiments on mouse tumor models.

[0440] Cells used in any of the above in vitro assays are primary cells or cell lines, including cells that naturally express or overexpress BCMA (e.g., human or monkey (e.g., cynomolgus monkey)) or GPRC5D (e.g., human or monkey (e.g., cynomolgus monkey) GPRC5D), such as cells that overexpress BCMA or GPRC5D, such as BCMA and GPRC5D positive cells, such as NCI-H929 or MM.1R or MOLP-8.

[0441] It will be appreciated that any of the above assays can be performed using a combination of the antibodies of the invention and additional active agents.

[0442] IX. Immunoconjugates, Pharmaceutical Compositions, Pharmaceutical Combination Products, and Kits of the Anti-BCMA Antibodies or Multispecific Antibodies of the Invention

[0443] In some embodiments, the present invention provides an immunoconjugate comprising any anti-BCMA antibody or multispecific antibody described herein. Preferably, the immunoconjugate comprises one or more other therapeutic agents (e.g., cytotoxins or small molecule compounds) or markers.

[0444] In some embodiments, the present invention provides a composition, a medicament, or a formulation comprising any of the anti-BCMA antibodies or multispecific antibodies described herein, preferably a composition is a pharmaceutical composition.

[0445] In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an anti-BCMA antibody or multispecific antibody of the invention in combination with one or more other therapeutic agents.

[0446] The composition, medicine or preparation of the present invention may further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0447] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0448] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0449] The compositions, medicaments or preparations of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injections or eye drops), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0450] Medicaments or formulations comprising the anti-BCMA antibodies or multispecific antibodies described herein can be prepared by mixing an anti-BCMA antibody or multispecific antibody of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, for example, in the form of a lyophilized formulation or an aqueous solution.

[0451] The compositions or medicaments or formulations of the present invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.

[0452] The present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-BCMA antibody or multispecific antibody of the present invention and one or more other therapeutic agents.

[0453] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0454] - a first container containing a pharmaceutical composition comprising an anti-BCMA antibody or multispecific antibody of the present invention;

[0455] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0456] In some embodiments, the additional therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0457] X. Uses of anti-BCMA antibodies or multispecific antibodies and methods of using the same

[0458] In one aspect, the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an anti-BCMA antibody or multispecific antibody of the present invention, or an immunoconjugate, composition, or drug or formulation comprising the same. In some embodiments, the present invention provides a method for specifically activating T cells in a subject, comprising administering to the subject an antibody or multispecific antibody of the present invention, or an immunoconjugate, composition, or drug or formulation comprising the same.

[0459] In some embodiments, the disease is, for example, a tumor, such as cancer. The cancer can be in the early, middle, or late stages or be metastatic. In some embodiments, the tumor can be a solid tumor or a hematological tumor. In some embodiments, the tumor is a myeloma, such as multiple myeloma, such as metastatic multiple myeloma, colon cancer, rectal cancer, or colorectal cancer.

[0460] In some embodiments, the disease treatment would benefit from activation of the CD3 signaling pathway and / or activation of T cells.

[0461] In some embodiments, the tumor or cancer is a BCMA-positive tumor or cancer. In some embodiments, the BCMA-positive tumor or cancer is characterized as a tumor or cancer that has or has elevated protein and / or nucleic acid levels of BCMA in a patient (e.g., in a patient's tumor or cancer tissue or cells) (e.g., compared to the protein and / or nucleic acid levels of BCMA in the same tissue of a healthy individual, or compared to the protein and / or nucleic acid levels of BCMA in adjacent healthy tissues of the patient), for example, the tumor or cancer has or has elevated protein and / or nucleic acid levels of BCMA in tumor cells (e.g., compared to the protein and / or nucleic acid levels of BCMA in cells of the same tissue of a healthy individual, or compared to the protein and / or nucleic acid levels of BCMA in healthy cells of the same tissue of the patient or adjacent healthy tissue).

[0462] In some embodiments, the tumor or cancer is a GRPC5D-positive tumor or cancer. In some embodiments, the GPRC5D-positive tumor or cancer is characterized as having or having elevated protein levels and / or nucleic acid levels of GPRC5D in a patient (e.g., in a patient's tumor or cancer tissue or cells) (e.g., compared to the protein levels and / or nucleic acid levels of GPRC5D in the same tissue of a healthy individual, or compared to the protein levels and / or nucleic acid levels of GPRC5D in the adjacent healthy tissue of the patient), or a tumor or cancer, for example, having or having elevated protein levels and / or nucleic acid levels of GPRC5D in tumor cells of the tumor or cancer (e.g., compared to the protein levels and / or nucleic acid levels of GPRC5D in cells of the same tissue of a healthy individual, or compared to the protein levels and / or nucleic acid levels of GPRC5D in healthy cells of the same tissue of the patient or in cells of adjacent healthy tissue).

[0463] In some embodiments, the tumor or cancer is a BCMA-positive and GPRC5D-positive tumor or cancer.

[0464] The anti-BCMA antibodies or multispecific antibodies of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration, intrapulmonary administration and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion. Depending to some extent on whether the medication is short-term or long-term, the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0465] For the prevention or treatment of disease, the appropriate dosage of the anti-BCMA antibodies or multispecific antibodies of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments. In other aspects, the present invention provides the use of the anti-BCMA antibodies or multispecific antibodies of the invention, or immunoconjugates or compositions or combination products comprising the same, in the manufacture or preparation of a medicament for the uses described herein, e.g., for the prevention or treatment of the relevant diseases or conditions mentioned herein.

[0466] In some embodiments, the anti-BCMA antibodies or multispecific antibodies (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0467] In some embodiments, the treatment modality is, for example, surgery or radiation therapy.

[0468] In some embodiments, the additional therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0469] XI. Diagnosis and Detection

[0470] In one aspect, the present invention also relates to methods for diagnosis and detection (e.g., for diagnostic or non-diagnostic purposes) of anti-BCMA antibodies or multispecific antibodies and compositions for diagnosis and detection comprising the same.

[0471] In certain embodiments, the anti-BCMA antibodies provided herein can be used to detect the presence of BCMA in a biological sample. In certain embodiments, the bispecific antibodies provided herein can be used to detect the presence of CD3 and / or BCMA in a biological sample. In certain embodiments, the trispecific antibodies provided herein can be used to detect the presence of CD3 and / or BCMA and / or GPRC5D in a biological sample.

[0472] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid, such as blood, serum, or plasma.

[0473] In certain embodiments, the method comprises contacting a biological sample with an anti-BCMA antibody or multispecific antibody as described herein under conditions that allow it to bind to BCMA, and detecting whether a complex is formed between the anti-BCMA antibody or multispecific antibody and BCMA. The formation of a complex indicates the presence of BCMA. The method can be an in vitro or in vivo method.

[0474] In certain embodiments, labeled anti-BCMA antibodies or multispecific antibodies are provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected moieties such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction. In some embodiments, the label is, for example, a marker such as biotin or hFc.

[0475] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-BCMA antibody or multispecific antibody of the invention. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during or after treatment with another therapy.

[0476] In some embodiments, BCMA and / or GPRC5D is detected prior to treatment, e.g., prior to initiation of treatment or prior to a treatment after a treatment interval.

[0477] XII. Specific Implementation Plan

[0478] In some aspects, the present invention relates to the following specific embodiments:

[0479] 1. A VHH antibody that specifically binds to BCMA, comprising

[0480] three complementarity determining regions (CDRs) contained in the VHH represented by any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23,

[0481] Preferably, the CDR sequences are defined according to IMGT.

[0482] 2. The VHH antibody of embodiment 1, comprising the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0483] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 9 or 13, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0484] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 5, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 6, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

[0485] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0486] 3. The VHH antibody of embodiment 1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0487] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23; or

[0488] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23; or

[0489] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22, or 23, preferably, the amino acid changes do not occur in the CDR regions.

[0490] 4. A heavy chain antibody that specifically binds to BCMA, comprising the VHH antibody of any one of embodiments 1-3.

[0491] 5. The heavy chain antibody of embodiment 4, comprising the VHH antibody of any one of embodiments 1-3 connected to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3 or human IgG4, optionally, the VHH antibody is connected to the Fc region via a hinge region or a portion thereof, optionally, the amino acid sequence of the hinge region portion is EPKSS (SEQ ID NO: 94) or EPKSC (SEQ ID NO: 96).

[0492] 6. The heavy chain antibody of embodiment 4, comprising the VHH antibody of any one of embodiments 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1, IgG2, IgG3 or IgG4, optionally comprising L234A / L235A mutations, D265A mutations and P329A mutations, preferably, the Fc region

[0493] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 80, 90, 97 or 98; or

[0494] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, 90, 97 or 98; or

[0495] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 80, 90, 97, or 98.

[0496] 7. The heavy chain antibody according to embodiment 4, wherein

[0497] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 44-46 and 48-55; or

[0498] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 44-46 and 48-55; or

[0499] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 44 to 46 and 48 to 55, preferably, the amino acid changes do not occur in the CDR regions.

[0500] 8. The VHH antibody of any one of embodiments 1-3, or the heavy chain antibody of any one of embodiments 4-7, wherein the antibody is a chimeric antibody or a humanized antibody.

[0501] 9. A multispecific antibody comprising a first antigen-binding region and a second antigen-binding region, and optionally a third antigen-binding region, wherein the second antigen-binding region specifically binds to BCMA, and comprises the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8. Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0502] 10. The multispecific antibody of embodiment 9, wherein the first antigen binding region specifically binds to CD3.

[0503] 11. The multispecific antibody of embodiment 10, wherein the first antigen-binding region comprises a VH and a VL, the VH comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, namely, HCDR1, HCDR2, and HCDR3, and the VL comprises three complementarity determining regions (LCDRs) from a light chain variable region, namely, LCDR1, LCDR2, and LCDR3, wherein (i) HCDR1, HCDR2, and HCDR3 are the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 34, and LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 35; or (ii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 28, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 29, HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 30, LCDR1 consists of the amino acid sequence shown in SEQ ID NO: 31, and LCDR2 consists of the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 32 is composed of LCDR3, and the amino acid sequence shown in SEQ ID NO: 33 is composed of LCDR4.

[0504] 12. The multispecific antibody of embodiment 11, wherein the first antigen-binding region comprises a VH and a VL, wherein the VH comprises or consists of an amino acid sequence as shown in SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or the VL comprises or consists of an amino acid sequence as shown in SEQ ID NO: 35, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0505] 13. The multispecific antibody according to any one of embodiments 10-12, wherein the first antigen binding region is a Fab or scFv that specifically binds to CD3.

[0506] 14. The multispecific antibody according to embodiment 13, wherein the Fab comprises VH and CH1 of the first antigen binding region, and optionally a portion of the hinge region (e.g., EPKSS and EPKSC), wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

[0507] 15. The multispecific antibody of embodiment 14, wherein the CH1

[0508] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 88; or

[0509] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 88.

[0510] 16. The multispecific antibody of embodiment 15, wherein the Fab heavy chain of the first antigen binding region

[0511] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 11;

[0512] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 11; or

[0513] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 11;

[0514] and / or the Fab light chain of the first antigen binding region

[0515] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 60;

[0516] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 60; or

[0517] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 60.

[0518] 17. The multispecific antibody of embodiment 13, wherein the scFv comprises a heavy chain variable region VH comprising a first antigen binding region from N-terminus to C-terminus, a linker and a light chain variable region of the first antigen binding region.

[0519] 18. The multispecific antibody of embodiment 17, wherein the scFv comprises the sequence shown in SEQ ID NO: 109 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 109 and having the same CDRs.

[0520] 19. The multispecific antibody of any one of embodiments 9-18, wherein the second antigen binding region is the VHH of any one of embodiments 1-3 and 8.

[0521] 20. The multispecific antibody of any one of embodiments 9-19, wherein the multispecific antibody is a trispecific antibody and comprises a third antigen binding region that specifically binds to GPRC5D.

[0522] 21. The multispecific antibody of embodiment 20, wherein the third antigen binding region comprises or consists of an anti-GPRC5D VHH.

[0523] 22. The multispecific antibody of embodiment 21, wherein the anti-GPRC5D VHH comprises or consists of a heavy chain variable region comprising

[0524] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NO: 27 or 106, or

[0525] (ii) Complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 26.

[0526] 23. The multispecific antibody of embodiment 22, wherein the heavy chain variable region

[0527] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 27 or 106; or

[0528] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 27 or 106; or

[0529] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 27 or 106, preferably, the amino acid changes do not occur in the CDR regions.

[0530] 24. The multispecific antibody of embodiment 20, wherein the third antigen-binding region comprises a VH and a VL, wherein the VH comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL comprises three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein

[0531] (i) HCDR1, HCDR2, and HCDR3 are the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH set forth in SEQ ID NO: 42, and LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL set forth in SEQ ID NO: 43; or

[0532] (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 39, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 40, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 41.

[0533] 25. The multispecific antibody of embodiment 24, wherein the third antigen-binding region comprises VH and VL, wherein

[0534] The VH comprises or consists of the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or

[0535] The VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0536] 26. The multispecific antibody of embodiment 24 or 25, wherein the third antigen binding region is a Fab that specifically binds to GPRC5D.

[0537] 27. The multispecific antibody of embodiment 26, wherein the Fab comprises VH and CH1 of the third antigen binding region, and optionally the CH1 comprises a partial hinge region (e.g., EPKSS and EPKSC), wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

[0538] 28. The multispecific antibody of embodiment 27, wherein the CH1

[0539] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 88; or

[0540] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 88.

[0541] 29. The multispecific antibody of embodiment 28, wherein the Fab heavy chain of the third antigen binding region

[0542] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 110;

[0543] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 110; or

[0544] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 110;

[0545] and / or the third antigen binding region of the Fab light chain

[0546] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 62;

[0547] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 62; or

[0548] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 62.

[0549] 30. The multispecific antibody of any one of embodiments 9-29, wherein the multispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different.

[0550] 31. The multispecific antibody of embodiment 30, wherein the two Fc regions are different, preferably, corresponding knob mutations and hole mutations are introduced into the two Fc regions, respectively.

[0551] 32. The multispecific antibody of embodiment 31, wherein

[0552] a) one Fc-region polypeptide comprises the knob mutation T366W and the other Fc-region polypeptide comprises the hole mutations T366S, L368A and Y407V, or

[0553] b) one Fc-region polypeptide comprises the knob mutations T366W and Y349C, and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V, and S354C, or

[0554] c) one Fc-region polypeptide comprises knob mutations T366W and S354C, and the other Fc-region polypeptide comprises hole mutations T366S, L368A, Y407V, and Y349C;

[0555] Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, eg, one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; eg, a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0556] 33. The bispecific antibody of any one of embodiments 30-32, wherein one or both of the Fc regions comprise a hinge region, e.g., EPKSS (SEQ ID NO: 94) or EPKSC (SEQ ID NO: 96).

[0557] 34. The multispecific antibody of embodiment 32, wherein

[0558] (i) Fc region containing knob mutation

[0559] a) comprises or consists of the amino acid sequence of SEQ ID NO: 47, 86 or 87; or

[0560] b) comprising or consisting of an amino acid sequence that is at least 90% identical, such as 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 47, 86 or 87 and comprises a knob mutation (e.g., S354C and T366W); and / or

[0561] (ii) Fc region containing hole mutation

[0562] a) comprises or consists of the amino acid sequence of SEQ ID NO: 84, 85 or 111; or

[0563] b) comprises or consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 84, 85 or 111, such as 95%, 96%, 97%, 99% or more identical and comprises hole mutations (e.g., Y349C, T366S, L368A and Y407V).

[0564] 35. The multispecific antibody of any one of embodiments 9-34, which is a bispecific antibody and comprises a first antigen-binding region, a second antigen-binding region and an Fc dimer, wherein the first antigen-binding region is a scFv fragment that specifically binds to CD3, and the second antigen-binding region is a VHH that specifically binds to BCMA, such as the VHH of any one of embodiments 1-3 and 8.

[0565] 36. The multispecific antibody of embodiment 35, comprising one or two VHHs that specifically bind to BCMA, an scFv of an anti-CD3 antibody, and an Fc heterodimer, wherein

[0566] The scFv comprises VH-VL (optionally, the VH and VL are fused via a linker), the VHH comprises or consists of a heavy chain variable region,

[0567] wherein the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the first Fc region (eg, comprising a knob mutation or a hole mutation) to constitute the first heavy chain;

[0568] The C-terminus of the one anti-BCMA VHH or two tandem anti-BCMA VHHs is fused to a second Fc region (eg, comprising a hole mutation or a knob mutation) to form a second heavy chain (eg, the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region).

[0569] 37. The multispecific antibody of embodiment 36, wherein the

[0570] The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0571] The second heavy chain comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 56, 102, 103 or 104, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0572] 38. The multispecific antibody of any one of embodiments 9-34, which is a trispecific antibody and comprises a first antigen-binding region, a second antigen-binding region, a third antigen-binding region and an Fc dimer, wherein the first antigen-binding region is a scFv fragment or a Fab fragment that specifically binds to CD3, the second antigen-binding region is a VHH that specifically binds to BCMA, such as the VHH of any one of embodiments 1-3 and 8, and the third antigen-binding region is a VHH or Fab fragment that specifically binds to GPRC5D.

[0573] 39. The multispecific antibody of embodiment 38, comprising an anti-GPRC5D VHH, an anti-BCMA VHH, an anti-CD3 Fab fragment, and an Fc dimer, wherein

[0574] The Fab fragment comprises VH-CH1 and VL-CL, the VHH comprises a heavy chain variable region, the trispecific antibody comprises a first heavy chain, a second heavy chain and a light chain, or consists of said chains, wherein

[0575] The first heavy chain comprises, from N-terminus to C-terminus, anti-GPRC5D VHH, anti-BCMA VHH, and the first Fc region fused via or without a linker; or anti-BCMA VHH, anti-GPRC5D VHH, and the first Fc region fused via or without a linker.

[0576] The second heavy chain comprises, from N-terminus to C-terminus, an anti-CD3 Fab fragment heavy chain and a second Fc region fused with or without a linker;

[0577] Light chain: anti-CD3 Fab fragment light chain.

[0578] 40. The multispecific antibody of embodiment 39, wherein

[0579] the first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;

[0580] The second heavy chain comprises, or consists of, the amino acid sequence of any one of SEQ ID NO: 59, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and / or

[0581] The light chain comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:60, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0582] 41. The multispecific antibody of embodiment 38, comprising an anti-GPRC5D Fab fragment, an anti-BCMA VHH, an anti-CD3 scFv, and an Fc dimer, wherein

[0583] The Fab fragment comprises VH-CH1 and VL-CL, the VHH comprises a heavy chain variable region, the scFV comprises VH-VL, and the trispecific antibody comprises a first heavy chain, a second heavy chain and a light chain, or consists of said chains, wherein

[0584] The first heavy chain comprises, from N-terminus to C-terminus, an anti-BCMA VHH and a first Fc region fused with or without a linker;

[0585] The second heavy chain comprises, from N-terminus to C-terminus, an anti-GPRC5D Fab fragment heavy chain, an anti-CD3 scFv, and a second Fc region fused via a linker or not;

[0586] Light chain: light chain of Fab fragment against GPRC5D.

[0587] 42. The multispecific antibody of embodiment 41, wherein

[0588] The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:56, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;

[0589] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0590] The light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0591] 43. A nucleic acid molecule encoding the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or any one chain of the multispecific antibody of any one of embodiments 9-42, or consisting of said nucleic acid sequence.

[0592] 44. An expression vector comprising the nucleic acid molecule of embodiment 43.

[0593] 45. A host cell comprising the nucleic acid molecule of embodiment 43 or the expression vector of embodiment 44, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells.

[0594] 46. ​​A method for preparing the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the multispecific antibody of any one of embodiments 9-42, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 43 or the expression vector of embodiment 44 under conditions suitable for expression of the chains of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0595] 47. An immunoconjugate comprising the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the multispecific antibody of any one of embodiments 9-42.

[0596] 48. A pharmaceutical composition, medicament or formulation comprising the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the multispecific antibody of any one of embodiments 9-42, or the immunoconjugate of embodiment 47, and optionally a pharmaceutically acceptable excipient.

[0597] 49. A pharmaceutical combination product comprising the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the multispecific antibody of any one of embodiments 9-42, or the immunoconjugate of embodiment 47, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

[0598] 50. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the bispecific antibody of any one of embodiments 9-42, or the immunoconjugate of embodiment 47, or the pharmaceutical composition or drug or formulation of embodiment 48; or the pharmaceutical combination product of embodiment 49.

[0599] 51. The method of embodiment 50, wherein the tumor cells of the cancer have elevated protein levels and / or nucleic acid levels of BCMA (e.g., elevated expression).

[0600] 52. The method of embodiment 50 or 51, wherein the cancer is a solid tumor or a hematological tumor, such as a myeloma, for example, multiple myeloma such as metastatic multiple myeloma, colon cancer, rectal cancer, or colorectal cancer.

[0601] 53. The method of any one of embodiments 50-52, wherein the method further comprises administering the drug in combination with other therapies, such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

[0602] 54. A method for detecting the presence of BCMA in a biological sample, comprising:

[0603] (i) contacting a biological sample with the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the multispecific antibody of any one of embodiments 9-42 under conditions that allow binding thereof to BCMA,

[0604] (ii) detecting whether a complex is formed between the antibody or bispecific antibody and BCMA,

[0605] The formation of the complex indicates the presence of BCMA. Example

[0606] Example 1: Production of BCMA antibodies by alpaca immunization

[0607] 1.1 Alpaca immune library construction and screening

[0608] 1.1.1 Animal immunization

[0609] Two alpacas (Chengdu Apak Biotechnology Co., Ltd.) were immunized with a mixture of human BCMA recombinant protein (ACRO, Catalog No.: BCA-H522y) and cynomolgus monkey BCMA-human IgG1Fc recombinant protein (ACRO, Catalog No.: BCA-C5253) at a volume ratio of 1:1 (equal concentrations, 0.5 mg each). The alpacas were immunized once every 21 days for four injections (Table 1). Venous blood was collected from the alpacas on the 10th day after the third and fourth immunizations, respectively. The alpaca immune serum was assayed by ELISA using human and cynomolgus monkey BCMA recombinant proteins, and one alpacas was selected for blood collection and library construction.

[0610] Table 1. Alpaca immunization schedule

[0611] 1.1.2 Construction and screening of alpaca immune library

[0612] Total RNA was extracted from alpaca peripheral blood using the Trizol method and reverse transcribed using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A) to obtain cDNA. Nested PCR amplification of the cDNA was performed to recover a 750 bp product containing the nanobody, which was then purified by a second round of PCR amplification. The VHHs were then ligated into phage vectors using enzyme digestion and ligation, followed by electroporation, transformation, and plate plating. The next day, the library capacity was determined to be 4.49 × 10e9 CFU, with an insertion rate of 100%. After two rounds of solid-phase screening with human BCMA recombinant protein, positive clones were identified by sequencing. The partial amino acid sequences obtained are shown in Table 2.

[0613] Table 2. Amino acid sequences of camel-derived VHHs of anti-BCMA antibodies

[0614] Example 2: Characterization of anti-human BCMA chimeric antibodies

[0615] 2.1 Synthesis and expression of anti-human BCMA chimeric antibodies

[0616] The amino acid sequence of the camel-derived antibody VHH in Table 2 was added to the hinge region (EPKSS (SEQ ID NO: 94)) and then inserted into the expression vector pcDNA3.1(+) containing the human Fc constant region (SEQ ID NO: 90) to obtain plasmids encoding each chimeric antibody.

[0617] The specific chimeric antibody sequences obtained are as follows:

[0618] Plasmids encoding each anti-BCMA antibody were transfected into ExpiCHO-S cells to express camelid chimeric antibodies. 269B094 (a fully human monoclonal antibody against BCMA) (heavy chain amino acid sequence SEQ ID NO: 76) was used as a control antibody, the sequence of which is from CN2017 / 096938. Similarly, the control antibody was expressed. The antibodies were purified accordingly.

[0619] Specifically, use ExpiCHO according to the manufacturer's product manual. TMExpiCHO-S cells were transfected with a plasmid encoding an anti-human BCMA chimeric antibody using an expression system (ThermoFisher, Cat# A29133). A control antibody, 269B094, was similarly expressed. Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the expressed and secreted antibody in the supernatant was purified using a MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibody was concentrated and sterile filtered. The purity of the antibody protein was determined by SDS-PAGE and size exclusion. The results showed that the antibody was >90% pure and ready for use in the next step.

[0620] 2.2 Binding of anti-human BCMA chimeric antibodies to engineered cells expressing human BCMA protein

[0621] The sequence encoding human BCMA (Accession # Q02223-1) was cloned into the PiggyBac Dual promoter (SBI, Catalog No. PB513-B1) expression vector and transfected into CHO-K1 cells (ATCC, Catalog No. CCL-61) by electroporation. TM ), and then screened with 4 μg / mL puromycin (Gibco, catalog number: A1113802) to obtain CHO-K1 cells that highly express human BCMA (hereinafter also referred to as: CHO-K1-huBCMA).

[0622] A cell binding experiment was performed to determine whether the anti-BCMA humanized antibody of the present invention could bind to the human BCMA protein stably expressed on the cell surface.

[0623] After enzymatic digestion of the CHOK1-huBCMA cell line, a single-cell suspension of CHOK1-huBCMA cells was obtained and centrifuged at 400 × g at room temperature, after which the medium was discarded. The cell pellet was washed once with PBS and resuspended in a serial dilution of anti-human BCMA chimeric antibody and control antibody 269B094 (initial concentration: 20 μg / mL, 4-fold serial dilutions, totaling 8 concentration points) and incubated at 4°C for 30 minutes. The cells were washed once with PBS and then incubated at 4°C for 30 minutes with a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098). The cells were then washed twice with PBS and resuspended. Fluorescence signals were detected in two channels using a Beckman Cytoflex flow cytometer.

[0624] From the results shown in FIG2 , it can be seen that the binding activity of all the tested samples to CHOK1-huBCMA engineered cells expressing human BCMA protein is better than that of the control sample.

[0625] Example 3: Humanization and characterization of alpaca-derived anti-human BCMA antibodies

[0626] 3.1 Humanization of Alpaca-derived Anti-Human BCMA Antibody

[0627] The antibodies obtained in Example 2.1 were humanized. Specifically, the sequences of antibodies BH56 and HA68 were searched and aligned in the IMGT database. The human germline gene sequence IGHV3-53*03, which has high homology to the variable region of antibody BH56, and the human germline gene sequence IGHV3-53*02, which has high homology to the variable region of antibody HA68, were obtained as the humanized frameworks for the heavy chain variable regions. The CDRs from the heavy chain variable regions of antibodies BH56 and HA68 were transplanted into the corresponding humanized frameworks to form humanized antibodies. To maintain the affinity of the humanized antibodies for human BCMA, the obtained humanized antibody framework regions were backmutated.

[0628] The humanized heavy chain variable region sequence was thus obtained, and the humanized variable region amino acid sequence is listed in Table 3. The variable region amino acid sequence was sent to Shanghai Biointron Biological Inc. for codon optimization and gene synthesis.

[0629] Table 3. Heavy chain and variable region sequences of humanized antibodies

[0630] 3.2 Expression and purification of humanized anti-human BCMA antibodies

[0631] Similar to Example 2.1, plasmids encoding full-length anti-human BCMA humanized antibodies were transfected into ExpiCHO-S cells to express each anti-human BCMA humanized antibody, and the corresponding anti-human BCMA humanized antibodies were purified as described in Example 2.1. The purified anti-human BCMA humanized antibodies were concentrated and sterile filtered, and the purity of the anti-human BCMA humanized antibodies was determined by SDS-PAGE and size exclusion chromatography (SEC).

[0632] 3.3 Physicochemical Analysis of Humanized Anti-Human BCMA Antibodies

[0633] The purity of the obtained anti-human BCMA humanized antibodies was confirmed by size exclusion chromatography. Specifically, 20 μg of each humanized anti-human BCMA antibody sample listed in Table 3 was injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na2SO4 (pH 7.0) as the running buffer. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data was analyzed using OpenLAB software. Based on the results, humanized antibodies with an antibody purity of >90% were selected for the next step of the experiment.

[0634] 3.4 Physicochemical Analysis of Humanized Anti-Human BCMA Antibodies

[0635] In this experiment, the binding affinity of the humanized antibodies described above to human BCMA-His protein (ACRO, Catalog No. BCA-H522y) was measured using the ForteBio Octet RED96e according to the manufacturer's instructions. Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was pre-equilibrated in running buffer (1X PBS, XiGene, Catalog No. XG3650, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) for 10 minutes at room temperature. BCMA kinetics assays were performed in a 96-well plate as follows: a) Baseline equilibration with running buffer for 180 seconds; b) Antibody diluted in running buffer was added to a final concentration of 5 μg / mL and allowed to solidify for 200 seconds; c) Baseline equilibration with running buffer for 180 seconds; d) Human BCMA protein (100 nM) diluted in running buffer was added to each well at the following concentrations. Binding was allowed for 200 seconds, and dissociation was allowed for 180 seconds. e) Regeneration solution (0.01 M Gly-HCl, pH 1.5) was used for regeneration for 30 seconds. The experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software. The results are shown in the following table.

[0636] Table 4 Binding affinity of anti-human BCMA humanized antibodies to human BCMA protein

[0637] The results in Table 4 indicate that the humanized antibody binds to human BCMA protein with higher affinity than the chimeric antibody.

[0638] 3.5 Binding of anti-human BCMA humanized antibodies to tumor cell lines

[0639] A cell binding experiment was performed to determine whether the anti-BCMA humanized specific antibody of the present invention could bind to the human BCMA protein expressed on the surface of tumor cells; the target cells in the detection system were NCI-H929 (Nanjing Kebai Biotechnology Co., Ltd., CBP60243) tumor cells that endogenously express human BCMA.

[0640] NCI-H929 cells were harvested and centrifuged at 400 × g at room temperature, after which the medium was discarded. The cell pellet was washed once with PBS and resuspended in serially diluted anti-BCMA humanized antibody and 269B094 control antibody (initial concentration: 20 μg / mL, 4-fold serial dilutions, totaling 8 concentration points) and incubated at 4°C for 30 minutes. The cells were washed once with PBS and then incubated at 4°C for 30 minutes with a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098). The cells were then washed twice with PBS and resuspended. Fluorescence signals were detected in two channels using a Beckman Cytoflex flow cytometer.

[0641] As shown in the results in FIG3 , the binding activity of huHA68-3 to NCI-H929 cells was lower than that of HA68-IgG, but the binding activity was better than that of the control antibody.

[0642] Example 4 Preparation and Characterization of Anti-Human BCMA×CD3 Bispecific Antibody

[0643] 4.1 Bispecific Antibody Construction

[0644] The present invention constructs two bispecific antibodies with different asymmetric structures as shown in FIG1 , wherein the anti-human BCMA portion of each bispecific antibody is derived from the above-mentioned humanized antibodies huBH56-3 and huHA68-4, and the anti-human CD3 portion is derived from the humanized CD3 antibody sp34 (PCT / CN2023 / 071314). The constant regions of the bispecific antibodies all contain a knob-in-hole (KIH) structure (Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.). Such bispecific antibodies are also referred to herein as "anti-CD3 / BCMA bispecific antibodies" or "anti-CD3 / anti-BCMA bispecific antibodies," and sometimes referred to simply as "bispecific antibody molecules, bispecific antibodies." The anti-BCMA portion of the anti-CD3 / BCMA bispecific antibody targets cells expressing BCMA, while the anti-CD3 portion activates T cells. The simultaneous binding of bispecific antibodies to BCMA on tumor cells and CD3 on T cells helps to target tumor cells through activated T cells.

[0645] This application uses standard construction methods to obtain two anti-CD3 / BCMA bispecific antibodies with the structures shown in Figure 1:

[0646] huBH56-3-hole: The variable region sequence of the anti-BCMA humanized antibody huBH56-3 was cloned into the pcDNA3.1(+) vector containing Fc(hole) to express the huBH56-3-hole molecule.

[0647] 2xhuBH56-3-hole: PCR was used to amplify the sequence encoding the huBH56-3 variable region. Two sequences encoding the huBH56-3 variable region were then concatenated using overlapping PCR (with a connecting peptide GGGGS (SEQ ID NO: 99) between the two coding fragments). The concatenated sequence was then cloned into the pcDNA3.1(+) vector containing the Fc(hole) to express the resulting 2x huBH56-3-hole molecule.

[0648] huHA68-4-hole: The variable region sequence of the anti-BCMA humanized antibody huHA68-4 was cloned into the pcDNA3.1(+) vector containing Fc(hole) to express the huHA68-4-hole molecule.

[0649] 2xhuHA68-4-hole: PCR was used to amplify the sequence encoding the huHA68-4 variable region. Overlapping PCR was then used to concatenate the two sequences encoding the huHA68-4 variable region (with a linker GGGGS between the two coding fragments). The concatenated sequence was then cloned into the pcDNA3.1(+) vector containing the Fc(hole) to express the resulting 2xhuHA68-4-hole molecule.

[0650] CD3 ScFv-knob: The nucleotide sequence encoding the anti-CD3 single-chain antibody 23L2 was cloned into the pcDNA3.1(+) vector with Fc(knob) to express the CD3 ScFv-knob molecule.

[0651] Teclistamab (a fully human bispecific antibody against BCMA / CD3) (heavy chain amino acid sequences are SEQ ID NOs: 63 and 65; light chain amino acid sequences are SEQ ID NOs: 64 and 66) was used as a control antibody. The sequences were obtained from US2017 / 0051068A1. The control antibody was similarly expressed and the antibody was purified accordingly.

[0652] According to the specific composition shown in Table 5, the above-mentioned expression vectors were combined accordingly and expressed under appropriate conditions to obtain the bispecific antibody in Figure 1.

[0653] The steps for construction, expression, purification, and preliminary analysis of the bispecific antibody were the same as those in Example 2.1.

[0654] Table 5. Humanized bispecific antibodies

[0655] The obtained heavy chain and light chain expression vectors (see Table 5) were co-transfected into ExpiCHO-S cells, and the bispecific antibody protein was expressed under appropriate conditions. The expression, purification, and preliminary analysis steps were the same as in Example 2.1.

[0656] 4.2 Binding of Anti-Human BCMA × CD3 Dual Antibody to Human BCMA Protein-Expressing Engineered Cells

[0657] A cell binding experiment was performed to determine whether the anti-BCMA×CD3 humanized bispecific antibody of the present invention could bind to the human BCMA protein stably expressed on the cell surface.

[0658] After enzymatic digestion of the CHOK1-huBCMA cell line, a single-cell suspension of CHOK1-huBCMA cells was obtained and centrifuged at 400 × g at room temperature, after which the medium was discarded. The cell pellet was washed once with PBS and resuspended in a serial dilution series of anti-BCMA humanized antibodies and a teclistamab analog control antibody (initial concentration: 20 μg / mL, 4-fold serial dilutions for a total of 8 concentration points) and incubated at 4°C for 30 minutes. The cells were washed once with PBS and then incubated with a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat#109-116-098), at 4°C for 30 minutes in the dark. The cells were washed twice with PBS and resuspended. Fluorescence signals were detected in two channels using a Beckman Cytoflex flow cytometer.

[0659] From the results shown in FIG4 , it can be seen that the binding activities of all tested samples to the engineered cells are better than those of the control sample.

[0660] 4.3 Cytotoxicity of anti-BCMA×CD3 bispecific antibodies against tumor cells

[0661] To examine the activity of anti-CD3 / BCMA bispecific antibodies in mediating T cell-mediated tumor cell cytotoxicity, we established a primary T cell-dependent cytotoxicity assay system using RPMI-8226 (TCHu234, Cell Bank of the Chinese Academy of Sciences), NCI-H929 (CBP60243, Nanjing Kebai Biotechnology Co., Ltd.), and 293T (GNHu17, Cell Bank of the Chinese Academy of Sciences) cell lines that endogenously express human BCMA as target cells and human peripheral blood mononuclear cells (PBMCs) (obtained from healthy volunteers) as effector cells.

[0662] PBMC cells were revived and the cell density was adjusted to 5-10×10 cells using 1640 complete medium (supplemented with 10% FBS). 6 / mL, and activated overnight with IL-2 (Jiangsu Jinsili Pharmaceutical Co., Ltd.) at a final concentration of 100 IU / mL. Target cells, RPMI-8226, NCI-H929, and BCMA-negative 293T cells, were centrifuged at 400 × g for 5 min, and the supernatant discarded. Target cell density was adjusted to 2 × 10 cells / mL using MEM-α (Gibco, Cat#41061-029) assay buffer containing 1% FBS (Gibco, Cat#10099-141) and 100 IU / mL IL-2. 5 / mL, and 50 μL per well (ie, 10,000 cells per well) were plated into a 96-well plate (Corning, Cat#3599).

[0663] Prepare a 5-fold arithmetic dilution of the test antibody (maximum test concentration is 1 μg / mL, with 9 concentration points in a 5-fold arithmetic dilution). Add 50 μL to each well. Also set up controls for maximum target cell killing (adding 2% Triton 100 lysis buffer to target cells), minimum killing (adding test buffer to target cells), and natural killer (adding effector cells to target cells). Add 50 μL to each well. The final ratio of effector cells (E) to target cells (T) is 10:1. Incubate in a cell culture incubator for 24 hours. After the incubation, remove the experimental plate and centrifuge at 400×g for 3 minutes to allow all cells to sink to the bottom of the plate. Carefully pipette 50 μL of supernatant into a new 96-well plate and add 50 μL of LDH detection solution (Roche, Cat#11644793001) and incubate at room temperature. When the color changes, read the plate on an F50 microplate reader with a detection wavelength of 492 nm and a reference wavelength of 650 nm. Detection and analysis are performed when the OD492 value of the maximum killing well is between 0.4 and 1.0.

[0664] The results, shown in Figure 5, demonstrate that the anti-CD3 / BCMA bispecific antibody specifically induced T cells to kill RPMI-8226 and NCI-H929 tumor cells, with stronger killing activity than the control antibody. However, it had no killing effect on BCMA-negative 293T cells. Therefore, the bispecific antibody of the present invention exhibited no significant nonspecific killing activity.

[0665] 4.4 Anti-BCMA×CD3 bispecific antibody induces cytokine release after T cell activation

[0666] To examine the simultaneous induction of cytokine secretion by T cells mediated by the anti-BCMA × CD3 bispecific antibody against tumor cells, we established a primary T cell-dependent cytotoxicity assay using NCI-H929 cells endogenously expressing human BCMA and GPRC5D as target cells and human peripheral blood mononuclear cells (PBMCs) as effector cells. Cytotoxicity incubation was performed as described in 4.3. At the endpoint of cytotoxicity, NCI-H929 supernatants were collected and assayed for IL-6 and IFN-γ.

[0667] After the killing incubation, the experimental plate was removed and centrifuged at 500×g for 5 minutes to allow all cells to sink to the bottom of the plate. 100 μL of supernatant was carefully transferred to a new 96-well plate, and the levels of human IFNγ (detection kit: Cisbio, Cat#62HIFNGPEH), human TNFα (detection kit: R&D, Cat#DY210), and human IL-6 (detection kit: R&D, Cat#DY206) factors in the supernatant were detected.

[0668] The results, shown in Figure 6, demonstrate that the anti-BCMA×CD3 bispecific antibody can induce T cells to release human IFNγ (Figure 6) and human IL-6 (Figure 6) in the NCI-H929 cell system. This trend in factor release is consistent with the cytotoxicity results. The stronger the cytotoxicity, the higher the factor release.

[0669] Example 5: Preparation and validation of GPRC5D VHH antibodies

[0670] 5.1 Using the alpaca immunization method and phage library display, the following camel-derived GPRC5D VHH antibody VHH8 was obtained

[0671] The human GPRC5D protein (NP_061124.1 (NCBI) or sp|Q9NZD1 (Uniprot)) gene sequence was codon-optimized and gene synthesized by General Biosystems (Anhui) Co., Ltd. and cloned into a stable expression vector. TM The gene sequence encoding the human GPRC5D protein (SEQ ID NO: 108) was transfected into CHO-K1 (ATCC, Cat# CCL-61) and 293T (Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Cat# SCSP-502) cells using 2000 (Invitrogen, Cat# 11668019) transfection reagent. The transfected cells were selected with 0.3 μg / mL puromycin (Gibco, Cat# A1113802) and plated to generate CHO-K1 (CHO-K1-huGPRC5D) and 293T engineered monoclonal cells (293T-huGPRC5D) that highly express human GPRC5D.

[0672] Table 6: Variable region amino acid sequences of camel-derived anti-GPRC5D VHH antibodies

[0673] The DNA sequences of the VHH antibodies in Table 6 were codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. The VHH genes encoding the antibodies were inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region Fc (SEQ ID NO: 80) to obtain a camelid anti-GPRC5D VHH antibody plasmid containing the human IgG1 heavy chain constant region Fc. The amino acid sequence of the antibody VHH-Fc containing the human IgG1 heavy chain constant region Fc is shown in SEQ ID NO: 107.

[0674] The plasmid encoding the camel-derived anti-GPRC5D VHH-Fc antibody (containing the human IgG1 heavy chain constant region Fc) described in Table 6 was transfected into ExpiCHO-S cells to express the antibody and then purified accordingly (see Example 2.1 for specific methods). The purified antibody was concentrated and sterile filtered, and the purity of the antibody protein was determined by SDS-PAGE and size exclusion. The results showed that the antibody purity was >90%, meeting the requirements and suitable for use in the next experiment.

[0675] 5.2 Binding of Camel-derived Anti-GPRC5D VHH Antibodies to Human GPRC5D Protein or Rhesus Monkey GPRC5D Protein Expressed on Cell Surfaces

[0676] A cell binding experiment was performed to determine whether the camel-derived anti-GPRC5D VHH antibody of the present invention could bind to the human GPRC5D protein or the cynomolgus monkey GPRC5D protein stably expressed on the surface of 293T cells.

[0677] After enzymatic digestion, a single cell suspension of both cells was obtained, which was centrifuged at 400 × g at room temperature and the culture medium was discarded. The cell pellet was washed once with PBS and centrifuged to discard the supernatant. The cell pellet was resuspended in a gradient dilution of the camel-derived anti-GPRC5D VHH-Fc antibody obtained in this application (initial concentration was 25 μg / mL, 4-fold gradient dilution down to 6.25 μg / mL, 1.563 μg / mL, 0.391 μg / mL, 0.0977 μg / mL, 0.0244 μg / mL, 0.0061 μg / mL, and 0.00153 μg / mL, a total of 8 points) and incubated at 4°C for 30 minutes. After washing the cells once with PBS, add the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific (Jackson ImmunoResearch, Cat#109-116-098) at a dilution of 1:200 and incubate at 4°C in the dark for 30 minutes. Wash the cells twice with PBS and resuspend them. Fluorescence signals in the PE channel were detected using a Beckman CytoFlex flow cytometer (Beckman).

[0678] The results shown in Figures 21 and 22 show that the camel-derived anti-GPRC5D VHH-Fc antibody can bind to cells expressing human GPRC5D protein or cynomolgus monkey GPRC5D protein.

[0679] 5.3 Humanization of camel-derived anti-GPRC5D antibody

[0680] Camelid antibody 8 was humanized using the method described in Example 3.

[0681] Thus, a humanized VHH antibody 8H5 was obtained, whose VHH sequence is SEQ ID NO: 27, and HCDR1, HCDR2, and HCDR3 under the IMGT numbering scheme are SEQ ID NO: 24, 25, and 26, respectively. The variable region amino acid sequence was sent to Universal Biosystems (Anhui) Co., Ltd. for codon optimization and gene synthesis. The genes encoding the antibody VHH region were sequentially inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region Fc to obtain a plasmid expressing the full-length heavy chain VHH-Fc of the anti-GPRC5D humanized antibody. For specific steps, see 5.1. The amino acid sequence of the obtained VHH-Fc is shown in SEQ ID NO: 105.

[0682] The plasmid combination encoding the full-length heavy chain VHH-Fc of the GPRC5D humanized antibody was transfected into ExpiCHO-S cells to express the anti-GPRC5D humanized antibody and purified accordingly (see Example 2.1 for specific methods). The purified antibody was concentrated, sterile filtered, and the protein purity was determined by SDS-PAGE and size exclusion chromatography (SEC).

[0683] 5.4 Physicochemical Analysis of Humanized Anti-GPRC5D Antibodies

[0684] The purity of the obtained humanized anti-GPRC5D VHH-Fc antibody was confirmed by size exclusion chromatography. Specifically, 20 μg of sample was injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na2SO4 (pH 7.0) as the running buffer. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data were analyzed using OpenLAB software.

[0685] Table 7 Purity of humanized antibodies

[0686] 5.5 Binding of humanized anti-GPRC5D VHH-Fc antibody to human GPRC5D protein expressed on the cell surface

[0687] A cell binding experiment was performed to determine whether the humanized anti-GPRC5D VHH-Fc antibody of the present invention could bind to the human GPRC5D protein stably expressed on the surface of 293T cells.

[0688] After enzymatic digestion, a single-cell suspension of 293T-huGPRC5D cells (constructed as described in Example 5.1) was obtained and centrifuged at 400 × g at room temperature, after which the culture medium was discarded. The cell pellet was washed once with PBS and centrifuged, and the supernatant was discarded. The cell pellet was resuspended in a gradient dilution of the humanized GPRC5D VHH-Fc antibody obtained herein (initial concentration: 25 μg / mL, 4-fold gradient dilution down to 6.25 μg / mL, 1.563 μg / mL, 0.391 μg / mL, 0.0977 μg / mL, 0.0244 μg / mL, 0.0061 μg / mL, and 0.00153 μg / mL, a total of 8 points) and incubated at 4°C for 30 minutes. After washing the cells once with PBS, add the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific (Jackson ImmunoResearch, Cat#109-116-098) at a dilution of 1:200 and incubate at 4°C in the dark for 30 minutes. Wash the cells twice with PBS and resuspend them. Fluorescence signals in the PE channel were detected using a Beckman CytoFlex flow cytometer (Beckman).

[0689] The results shown in FIG23 show that the humanized GPRC5D VHH-Fc antibody can bind to cells expressing human GPRC5D protein, and some clones have better binding ability than the control antibody to cells.

[0690] 5.6 Binding assay of humanized anti-GPRC5D VHH antibody to 293T engineered cells expressing proteins of the same family:

[0691] 5.6.1 Construction and identification of 293T engineered cells expressing proteins of the same family:

[0692] Transfected cell lines of GPRC5A, GPRC5B, and GPRC5C were generated using standard methods for characterization studies (Table 8).

[0693] The human GPRC5A protein (Q8NFJ5, SEQ ID NO: 81) gene vector was constructed by Nanjing Weilizhibo Biotechnology Co., Ltd., using cDNA (Sino, Cat# HG12833 UT) as a template to amplify the GPRC5A protein target fragment and clone it into the stable vector PiggyBac. The human GPRC5B protein (Q9NZH0, SEQ ID NO: 82) gene sequence and human GPRC5C protein (Q9NQ84, SEQ ID NO: 83) were codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. for CHO cells and cloned into the stable vector PiggyBac. Lipofectamine was used to express the protein. TM Gene sequences encoding human GPRC5A, GPRC5B, and GPRC5C proteins were transfected into 293T cells (Cell Bank of the Chinese Academy of Sciences, Cat# SCSP-502) using Invitrogen 2000 (Invitrogen, Cat# 11668019). Transfected cells were pressure-selected with 0.3 μg / mL puromycin (Gibco, Cat# A1113802) for at least one week. The pressure-selected cells were enzymatically digested to a single-cell suspension and incubated with 10 μg / mL dilutions of the commercially available positive antibodies: Human GPRC5A Antibody (R&D, MAB5239), Human GPRC5B Antibody (R&D, MAB10253), and Human GPRC5C Antibody (R&D, MAB6594). After incubation at 4°C for 3 minutes, the cell pellet was washed once with PBS, centrifuged, and the supernatant discarded. The cell pellet was resuspended in a 1:200 dilution of R-Phycoerythrin AffiniPure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), FcγFragment Specific (min X Hu, Bov, Rb Sr Prot) (Jackson ImmunoResearch, 115-115-164) and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS and the positive rate of cells was determined by flow cytometry. Flow cytometry analysis showed that 293T engineered polyclonal cells expressing human GPRC5A, GPRC5B, and GPRC5C were obtained through transfection (Table 8).

[0694] Table 8: GPRC5A, GPRC5B and GPRC5C expressing cell lines

[0695] 5.6.2 Binding of Humanized Anti-GPRC5D VHH Antibodies to 293T Engineered Cells Expressing Homologous Proteins

[0696] The three 293T engineered cells expressing the same family of proteins (293T-humanGPRC5A, 293T-humanGPRC5B, and 293T-humanGPRC5C) were enzymatically digested to obtain single-cell suspensions. Anti-human GPRC5D VHH-Fc antibody and control antibody GC5B596 (HC: SEQ ID NO:67; LC: SEQ ID NO:68) were diluted in PBS to 20 μg / mL and 2 μg / mL, respectively. The cell pellets were resuspended and incubated at 4°C for 30 minutes. Cells without antibody were used as a blank control. After washing once with PBS, the cells were resuspended in R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (min X Bov, Hrs, Ms Sr Prot) at a dilution of 1:200 and incubated at 4°C in the dark for 30 minutes. After washing the cells twice with PBS and resuspending them in 100 μL of PBS, antibody binding to the cells was assayed on a Beckman CytoFlex flow cytometer. To ensure that the cells expressed the correct protein, flow cytometry was performed using the commercially available positive antibodies Human GPRC5A Antibody (R&D, MAB5239), Human GPRC5B Antibody (R&D, MAB10253), and Human GPRC5C Antibody (R&D, MAB6594) as described above for the same family protein-engineered 293T cell lines.

[0697] As shown in Figures 24, 25 and 26, the flow cytometry results show that the anti-human GPRC5D VHH antibody binds weakly to 293T engineered cells expressing the same family proteins GPRC5A, GPRC5B and GPRC5C.

[0698] Example 6 Preparation and Characterization of Anti-Human BCMA×GPPRC5D×CD3 Trispecific Antibody

[0699] 6.1 Trispecific Antibody Construction

[0700] The present invention constructs two trispecific antibodies with different asymmetric structures as shown in FIG7 , wherein the anti-human BCMA portion of each trispecific antibody is derived from the above-mentioned humanized antibody huBH56-3, the anti-GPRC5D portion is derived from the humanized antibodies hu8H5 and hu29H6 (wherein the hu29H6 antibody is derived from PCT / CN2023 / 071314), and the anti-CD3 portion is derived from the humanized CD3 antibody (PCT / CN2023 / 071314). The bispecific antibody constant region contains a knob-in-hole structure (KIH) (Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.). Such trispecific antibodies are also referred to herein as "anti-CD3 / BCMA / GPRC5D trispecific antibodies" or "anti-CD3 / anti-BCMA / anti-GPRC5D trispecific antibodies," and are sometimes referred to as "trispecific antibody molecules, triantibodies." The anti-BCMA portion of the anti-CD3 / BCMA / GPRC5D trispecific antibody targets cells expressing BCMA, the anti-GPRC5D portion targets cells expressing GPRC5D, or both the anti-BCMA and anti-GPRC5D portions target tumor cells expressing both BCMA and GPRC5D, while the anti-CD3 portion activates T cells. The simultaneous binding of the three antibodies to BCMA and GPRC5D on tumor cells and CD3 on T cells facilitates targeted killing of tumor cells by activated T cells.

[0701] This application used standard construction methods to obtain two anti-CD3 / BCMA / GPRC5D trispecific antibodies with the structures shown in Figure 7:

[0702] CD3Fab heavy chain-knob: The sequence encoding the heavy chain variable region of the anti-CD3 humanized monoclonal antibody was cloned into the pcDNA3.1(+) vector containing the IgG1 heavy chain constant region containing the Fc (knob) to express the CD3Fab heavy chain-knob molecule.

[0703] CD3Fab light chain: The sequence encoding the light chain variable region of the anti-CD3 humanized monoclonal antibody H34L-2 was cloned into the pcDNA3.1(+) vector containing the IgG1 light chain lamda constant region to express the CD3-LC molecule.

[0704] huBH56-3-hole: The variable region sequence of the anti-BCMA humanized antibody hu BH56-3 was cloned into the pcDNA3.1(+) vector containing Fc(hole) to express the hu BH56-3-hole molecule.

[0705] hu8H5-huBH56-3-hole: Using overlapping PCR, the sequences encoding the hu8H5 variable region and the huBH56-3 variable region were concatenated (with a linker GGSGG between the two coding segments). The concatenated sequences were then cloned into the pcDNA3.1(+) vector containing the Fc(hole) to express the resulting hu8H5-huBH56-3-hole molecule.

[0706] 29H6-CD3ScFv-knob: Using overlapping PCR technology, the nucleotide sequences encoding the heavy chain variable region sequence of the anti-GPRC5D antibody 29H6 and the anti-CD3 single-chain antibody 23L2 were sequentially spliced ​​in series (containing a linker: GGGGS between the two coding fragments). The spliced ​​sequence was then cloned into the pcDNA3.1(+) vector containing the Fc(knob) to express the 29H6-CD3ScFv-knob molecule.

[0707] 29H6Fab light chain: The light chain variable region sequence encoding 29H6 was cloned into the pcDNA3.1(+) vector containing the IgG1 light chain kappa constant region to express the 29H6Fab light chain molecule.

[0708] According to the specific composition shown in Table 9, the above-mentioned expression vectors were combined accordingly and expressed under appropriate conditions to obtain the trispecific antibody in Figure 7.

[0709] The construction, expression, purification, and preliminary analysis steps of the trispecific antibodies were the same as those in Example 2.1.

[0710] Table 9. Trispecific Antibodies

[0711] The obtained heavy chain and light chain expression vectors (see Table 9) were co-transfected into ExpiCHO-S cells, and trispecific antibody proteins were expressed under appropriate conditions. The expression, purification, and preliminary analysis steps were the same as in Example 2.1.

[0712] The IBI3003 trispecific antibody was prepared according to the method disclosed in WO2022 / 174813 / A1.

[0713] The BGCB491 trispecific antibody was prepared according to the method disclosed in WO2022 / 175255A2.

[0714] 6.2 Binding experiment of anti-human BCMA×GPPRC5×CD3 trispecific antibody to tumor cells endogenously expressing human BCMA and human GPRC5D proteins

[0715] Cell binding experiments were performed to determine whether the trispecific antibody of the present invention could bind to proteins on the surface of tumor cells that endogenously express human BCMA and human GPRC5D proteins.

[0716] Single-cell suspensions of MM.1R (Cell Bank of the Chinese Academy of Sciences, SCSP-5050) and NCI-H929 (Nanjing Kebai Biotechnology Co., Ltd., CBP60243) were collected and centrifuged at 400 × g at room temperature, after which the culture medium was discarded. The cell pellets were washed once with PBS and resuspended in a serial dilution series of anti-CD3 / BCMA / GPRC5D trispecific antibodies (initial concentration: 150 nM, 4-fold serial dilutions, totaling 8 concentrations) and incubated at 4°C for 30 minutes. The cells were washed once with PBS and then added with a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098), incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS and resuspended. Fluorescence signals were detected in two channels using a Beckman Cytoflex flow cytometer.

[0717] From the results shown in FIG8 , it can be seen that all the tested samples have good binding activity with MM.1R and NCI-H929 tumor cells.

[0718] 6.3 Detection of the Cytotoxicity of Anti-BCMA×GPPRC5D×CD3 Trispecific Antibody Against Tumor Cells and T Cell Activation and Exhaustion Indicators

[0719] To detect the activity of anti-BCMA×GPPRC5D×CD3 trispecific antibodies in mediating T cell-mediated tumor cell killing, we established a primary T cell-dependent cytotoxicity assay system, using MM.1R (high) (Cell Bank of the Chinese Academy of Sciences, SCSP-5050), NCI-H929 (medium) (Nanjing Kebai Biotechnology Co., Ltd., CBP60243), and MOLP-8 (low) (Nanjing Kebai Biotechnology Co., Ltd., CBP60243) tumor cells endogenously expressing different levels of human BCMA and GPRC5D as target cells, and human peripheral blood mononuclear cells (PBMCs) (obtained from healthy human blood) as effector cells.

[0720] PBMC cells were revived and the cell density was adjusted to 5-10×10 cells using 1640 complete medium (supplemented with 10% FBS). 6 / mL, and activated overnight with IL-2 (Jiangsu Jinsili Pharmaceutical Co., Ltd.) at a final concentration of 100 IU / mL. Target cells MM.1R, NCI-H929, and MOLP-8 were centrifuged at 400 × g for 5 min, and the supernatant discarded. The target cell density was adjusted to 4 × 10 cells / mL using MEM-α (Gibco, Cat#41061-029) assay buffer containing 1% FBS (Gibco, Cat#TCHu 44) and 100 IU / mL IL-2. 5 / mL, and 50 μL per well (ie, 20,000 cells per well) were plated into a 96-well plate (Corning, Cat#3599).

[0721] Prepare a 5-fold arithmetic dilution of the test antibody (maximum test concentration is 10 μg / mL, with 9 concentration points in a 5-fold arithmetic dilution). Add 50 μL to each well. Also set up controls for maximum target cell killing (adding 2% Triton 100 lysis buffer to target cells), minimum killing (adding test buffer to target cells), and natural killer (adding effector cells to target cells). Add 50 μL to each well. The final ratio of effector cells (E) to target cells (T) is 3:1. Incubate in a cell culture incubator for 48 hours. After the incubation, remove the experimental plate and centrifuge at 400×g for 3 minutes to allow all cells to sink to the bottom of the plate. Carefully pipette 50 μL of supernatant into a new 96-well plate and add 50 μL of LDH detection solution (Roche, Cat#11644793001) and incubate at room temperature. When the color changes, read the plate on an F50 microplate reader with a detection wavelength of 492 nm and a reference wavelength of 650 nm. Detection and analysis are performed when the OD492 value of the maximum killing well is between 0.4 and 1.0.

[0722] The results are shown in Figure 9, which show that the anti-BCMA×GPPRC5D×CD3 trispecific antibody can specifically induce T cells to kill tumor cells, and the killing activity is stronger than that of the control antibody.

[0723] To examine the effects of the anti-BCMA×GPPRC5D×CD3 trispecific antibody on T cell function in the TDCC killing system, we tested T cell expression of CD25 (Biolegend, Cat#302612), CD69 (Biolegend, Cat#310904), PD1 (Biolegend, Cat#329908), and TIM3 (Biolegend, Cat#345006), as well as T cell apoptosis (Biolegend, Cat#423114) in the killing experiment.

[0724] Resuspend the remaining cells in the killing assay plate with PBS and transfer them to a 96-well V-bottom plate (NEST, Taobao). Centrifuge at 500 × g for 5 minutes to collect the cells and discard the supernatant. Resuspend the cells in PBS containing the detection antibody. After incubation at 4°C for 30 minutes, remove the assay plate, centrifuge at 500 × g for 5 minutes, discard the supernatant, and resuspend the cells in PBS for flow cytometry analysis to test for changes in T cell activation (CD25 and CD69 expression), T cell failure (PD1 and TIM3 expression), and T cell apoptosis.

[0725] The results are shown in Figure 10. The results of T cell activation and exhaustion markers were generally consistent with the killing trend, indicating that the anti-BCMA×GPPRC5D×CD3 trispecific antibody activated T cells more strongly than the control antibody and also promoted T cell apoptosis.

[0726] 6.4 Anti-BCMA×GPPRC5D×CD3 trispecific antibody induces cytokine release after T cell activation

[0727] To examine the simultaneous induction of cytokine secretion by the anti-BCMA × GPPRC5D × CD3 trispecific antibody-mediated T cell-mediated tumor cell cytotoxicity, we established a primary T cell-dependent cytotoxicity assay system using MM.1R, NCI-H929, and MOLP-8 cells endogenously expressing human BCMA and GPPRC5D as target cells and human peripheral blood mononuclear cells (PBMCs) as effector cells. Cytotoxicity incubation was performed as described in 5.8. At the endpoint, the supernatant was collected and assayed for IL-6, TNF-α, and IFN-γ.

[0728] After the killing incubation, the experimental plate was removed and centrifuged at 500×g for 5 minutes to allow all cells to sink to the bottom of the plate. 100 μL of supernatant was carefully transferred to a new 96-well plate, and the levels of human IFNγ (detection kit: Cisbio, Cat#62HIFNGPEH), human TNFα (detection kit: R&D, Cat#DY210), and human IL-6 (detection kit: R&D, Cat#DY206) factors in the supernatant were detected.

[0729] The results, shown in Figure 11, demonstrate that the anti-BCMA×GPPRC5D×CD3 trispecific antibody induced T cells to release human IFNγ (Figure), human TNFα (Figure), and human IL-6 (Figure) in the MM.1R, NCI-H929, and MOLP-8 cell systems. The trends in factor release were consistent with the cytotoxicity results. Stronger cytotoxicity correlated with higher factor release.

[0730] 6.5 Comparison of the Cytotoxicity of the Anti-CD3×BCMA×GPRC5D Trispecific Antibody and the CD3×BCMA, CD3×GPRC5D Bispecific Antibody Combination on Tumor and Negative Cells

[0731] To compare the T cell-mediated tumor cell killing activity of the anti-CD3×BCMA×GPRC5D trispecific antibody and the teclistamab-talquetamab dual antibody combination, we established a primary T cell-dependent cytotoxicity assay system, using MOLP-8 (Nanjing Kebai Biotechnology Co., Ltd., CBP60243) tumor cells that endogenously express human BCMA and GPRC5D and 293T (Cell Bank of the Chinese Academy of Sciences, GNHu17) negative cell line as target cells, and human peripheral blood mononuclear cells (PBMCs) (obtained from healthy human blood) as effector cells.

[0732] PBMC cells were revived and the cell density was adjusted to 5-10×10 cells using 1640 complete medium (supplemented with 10% FBS). 6 / mL, add IL2 (Jiangsu Jinsili Pharmaceutical Co., Ltd.) at a final concentration of 100 IU / mL and activate overnight. Target cells MOLP-8 and 293T were centrifuged at 400 × g for 5 min and the supernatant was discarded. After washing once with PBS, CellTrace TM Cells were labeled with Violet (1E7 cells / μL) (Invitrogen, Cat#C34557) dye and incubated at 37°C for 15 min. An equal volume of serum was then added and the cells were terminated at 37°C for 5 min. The supernatant was removed after centrifugation at 400 g for 4 min, and the labeled target cell density was adjusted to 4 × 10 cells / μL with MEM-α (Gibco, Cat#41061-029) assay buffer containing 1% FBS (Gibco, Cat#TCHu 44) and 100 IU / mL IL-2. 5 / mL, and 50 μL per well (ie, 20,000 cells per well) were plated into a 96-well plate (Corning, Cat#3599).

[0733] Prepare a 5-fold serial dilution of the test antibody (maximum test concentration is 2 nM, with 8 concentration points in a 5-fold serial dilution) and add 50 μL to each well. Simultaneously, set up controls for maximum target cell killing (target cells with 2% Triton 100 lysis buffer), minimum killing (target cells with test buffer), and natural killer (target cells with effector cells) and add 50 μL to each well. The final ratio of effector cells (E) to target cells (T) is 3:1. Incubate in a cell culture incubator for 48 hours. After incubation, remove the assay plate and centrifuge at 400 × g for 3 minutes to allow all cells to settle to the bottom. Carefully aspirate 100 μL of the supernatant into a fresh 96-well plate and reserve for later use. Resuspend the remaining cells in PBS and transfer them to a 96-well V-bottom plate (NEST, Cat#701201). Centrifuge at 500 × g for 5 minutes to collect the cells and discard the supernatant. Resuspend the cells in PBS containing the detection antibody Fixable Viability Stain 780 (Bioscience, Cat# 565388) (1:2000 dilution). After incubation at 4°C for 30 minutes, remove the plate and centrifuge at 500 × g for 5 minutes. Discard the supernatant and resuspend the cells in PBS for flow cytometry analysis to determine target cell killing.

[0734] The results are shown in Figure 12, demonstrating that the anti-CD3 / BCMA / GPRC5D trispecific antibody-mediated T cell cytotoxicity was stronger than the combined cytotoxicity of the control antibodies Teclistamab and Talquetamab. Furthermore, the TriBH563CD3G111-1G4S trispecific antibody showed no nonspecific cytotoxicity against 293T-negative cells.

[0735] 6.6 Anti-CD3×BCMA×GPRC5D trispecific antibody and CD3×BCMA, CD3×GPRC5D bispecific antibody induce cytokine release after T cell activation on tumor cells and negative cells

[0736] To compare the effects of the anti-CD3×BCMA×GPRC5D trispecific antibody and the teclistamab / talquetamab dual antibody combination on T cell-mediated tumor cell cytotoxicity and cytokine secretion, we established a primary T cell-dependent cytotoxicity assay system. MOLP-8 (Nanjing Kebai Biotechnology Co., Ltd., CBP60243), a tumor cell line that endogenously expresses both human BCMA and GPRC5D, and a 293T (Cell Bank, Committee for Type Culture Collection, Chinese Academy of Sciences, GNHu17) negative cell line were used as target cells, and human peripheral blood mononuclear cells (PBMCs) (derived from healthy individuals) were used as effector cells. Cytotoxicity incubation was performed as described in Example 6.3. At the endpoint of cytotoxicity, the supernatant was collected and assayed for TNF-α.

[0737] After the killing incubation, the experimental plate was removed and centrifuged at 500×g for 5 min to allow all cells to sink to the bottom of the plate. 100 μL of supernatant was carefully pipetted into a new 96-well plate, and the level of human TNFα (detection kit: R&D, Cat#DY210) factor in the supernatant was detected.

[0738] The results, shown in Figure 13, show that the trend in factor release is consistent with the killing results. The stronger the killing effect, the higher the factor release. Furthermore, the TriBH563CD3G111-1G4S triple antibody did not significantly promote T cell factor release in the 293T negative cell killing assay.

[0739] 6.7 Binding Assay of Anti-CD3×BCMA×GPRC5D Trispecific Antibody to Tumor Cells Endogenously Expressing Human BCMA and Human GPRC5D Proteins

[0740] Cell binding experiments were performed to determine whether the trispecific antibody of the present invention could bind to proteins on the surface of tumor cells that endogenously express human BCMA and human GPRC5D proteins.

[0741] Single-cell suspensions of AMO-1 (Nanjing Kebai Biotechnology Co., Ltd., CBP60242) and ARD (Wuhan Punosai Life Science Co., Ltd., CL-0778) were collected and centrifuged at 400 × g at room temperature, after which the culture medium was discarded. The cell pellet was washed once with PBS and resuspended in a serially diluted anti-CD3 / BCMA / GPRC5D trispecific antibody (initial concentration: 200 nM, 5-fold serial dilutions, totaling 8 concentration points) and incubated at 4°C for 30 minutes. The cells were washed once with PBS and then incubated with a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098), at 4°C for 30 minutes in the dark. The cells were washed twice with PBS and resuspended. Fluorescence signals were detected in two channels using a Beckman Cytoflex flow cytometer.

[0742] From the results shown in Figure 14, it can be seen that the tested trispecific antibodies have good binding activity with both AMO-1 and ARD tumor cells, and the binding activity is stronger than that of the control bispecific antibodies Teclistamab, Talquetamab and the control tertiary antibodies IBI3003 and BGCB491.

[0743] 6.8 Cytotoxicity of anti-CD3×BCMA×GPRC5D trispecific antibody against tumor cells

[0744] To detect the activity of anti-CD3×BCMA×GPRC5D trispecific antibodies in mediating T cell-mediated tumor cell killing, we established a primary T cell-dependent cytotoxicity assay system, using AMO-1 (Nanjing Kebai Biotechnology Co., Ltd., CBP60242) and ARD (Wuhan Pronocell Life Science Co., Ltd., CL-0778) tumor cells endogenously expressing different levels of human BCMA and GPRC5D as target cells and human peripheral blood mononuclear cells (PBMCs) (obtained from healthy human blood) as effector cells.

[0745] PBMCs were revived and adjusted to a cell density of 5–10 × 10⁶ / mL using 1640 complete medium (supplemented with 10% FBS). IL-2 (Jiangsu Jinsili Pharmaceutical Co., Ltd.) was added to a final concentration of 100 IU / mL for activation overnight. For target AMO-1 and ARD cells, cells were harvested by centrifugation at 400 × g for 5 min and the supernatant discarded. After washing once with PBS, the cells were added with CellTrace TM Cells were labeled with Violet dye (1E7 cells / μL) (Invitrogen, Cat#C34557) and incubated at 37°C for 15 min. An equal volume of serum was then added and the cells were incubated at 37°C for 5 min. The supernatant was removed after centrifugation at 400 g for 4 min, and the labeled target cell density was adjusted to 4 × 105 cells / mL with MEM-α (Gibco, Cat#41061-029) assay buffer containing 1% FBS (Gibco, Cat#TCHu 44) and 100 IU / mL IL-2. 50 μL of the assay buffer (i.e., 20,000 cells / well) was plated into a 96-well plate (Corning, Cat#3599).

[0746] Prepare a 5-fold serial dilution of the test antibody (maximum test concentration of 0.5 nM, 8 concentration points in 5-fold serial dilutions) and add 50 μL to each well. Simultaneously, set up controls for maximum target cell killing (target cells with 2% Triton 100 lysis buffer), minimum killing (target cells with test buffer), and natural killer (target cells with effector cells) and add 50 μL to each well. The final ratio of effector cells (E) to target cells (T) was 3:1. Incubate in a cell culture incubator for an additional 48 hours. After incubation, remove the assay plate and centrifuge at 400 × g for 3 minutes to allow all cells to settle to the bottom. Carefully aspirate 100 μL of the supernatant into a new 96-well plate and reserve for later use. Resuspend the remaining cells in PBS and transfer the cells to a 96-well V-bottom plate (NEST, Taobao). Centrifuge at 500 × g for 5 minutes to collect the cells and discard the supernatant. Resuspend the cells in PBS containing the detection antibody Fixable Viability Stain 780 (Bioscience, Cat# 565388) (1:2000 dilution). After incubation at 4°C for 30 minutes, remove the plate and centrifuge at 500 × g for 5 minutes. Discard the supernatant and resuspend the cells in PBS for flow cytometry analysis to determine target cell killing.

[0747] The results are shown in Figure 15, indicating that the anti-CD3×BCMA×GPRC5D trispecific antibody can specifically induce T cells to kill tumor cells, among which Tri8H5BH563-Fab has stronger killing activity than the control dual antibodies Teclistamab, Talquetamab and the control triple antibodies IBI3003 and BGCB491.

[0748] 6.9 Anti-CD3×BCMA×GPRC5D trispecific antibody induces cytokine release after T cell activation

[0749] To examine the simultaneous induction of cytokine secretion by T cells mediated by the anti-CD3×BCMA×GPRC5D trispecific antibody against tumor cells, we established a primary T cell-dependent cytotoxicity assay using AMO-1 and ARD cells endogenously expressing human BCMA and GPRC5D as target cells and human peripheral blood mononuclear cells (PBMCs) as effector cells. Cytotoxicity incubations were performed as described in Supplementary Example 2. At the endpoint of cytotoxicity, the supernatant was assayed for TNF-α.

[0750] After the killing incubation, the experimental plate was removed and centrifuged at 500×g for 5 min to allow all cells to sink to the bottom of the plate. 100 μL of supernatant was carefully pipetted into a new 96-well plate, and the release level of human TNFα (detection kit: R&D, Cat#DY210) factor in the supernatant was detected.

[0751] The results, shown in Figures 17 and 18, demonstrate that the anti-CD3×BCMA×GPRC5D trispecific antibody can induce T cells to release human TNFα in both the AMO-1 and ARD systems (Figures 17 and 18). The trend in TNFα release is consistent with the cytotoxicity results. Stronger cytotoxicity correlates with higher TNFα release.

[0752] Example 7 In vivo efficacy of anti-BCMA×GPRC5D×CD3 trispecific antibody in the hPBMC-B-NDG mouse NCI-H929 tumor model

[0753] The in vivo antitumor effect of an anti-BCMA×GPRC5D×CD3 trispecific antibody was investigated in the hPBMC-B-NDG mouse NCI-H929 tumor model.

[0754] Severely immunodeficient mice B-NDG were purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.; human peripheral blood mononuclear cells (PBMCs) were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.

[0755] Human myeloma cells NCI-H929 were purchased from Nanjing Kebai Biotechnology Co., Ltd. The cells were cultured in an incubator at 37°C and 5% CO2 in RPMI 1640 medium containing 10% inactivated fetal bovine serum.

[0756] On Day 8, 18 severely immunodeficient B-NDG mice were inoculated with 5.5×10 6 Human peripheral blood mononuclear cells / 0.2 mL RPMI 1640 / mouse, and then on Day 0, 5 × 10 6 NCI-H929 cells / 0.2mL RPMI 1640+Matrigel / mouse. When the average tumor volume reached about 147.7mm3, 12 suitable mice were selected for enrollment based on the mouse tumor volume and PBMC reconstruction (hCD45% proportion) and randomly assigned to two experimental groups, with 6 mice in each group. On days 0, 3, 7, and 10 after grouping, blank control PBS and Talquetamab (0.3mg / kg) and Tri8H5BH563-Fab trispecific antibody (0.3mg / kg) were intraperitoneally administered to the mice, respectively. Tumor volume was monitored by vernier caliper measurement twice a week during the experiment.

[0757] Treatment with the anti-GPRC5D×CD3 bispecific antibody Talquetamab resulted in nonsignificant inhibition of tumor growth compared to the PBS group, with a TGI of 37%. Treatment with the anti-BCMA×GPRC5D×CD3 trispecific antibody Tri8H5BH563-Fab resulted in significant inhibition of tumor growth compared to the PBS group, with a TGI of 80.4%. The results are shown in Figure 19 and Table 7 below.

[0758] Table 10. Inhibition of NCI-H929 tumor growth by the test article

[0759] Note: a. Mean ± standard error; b. TGI = (1 - change in tumor volume of the treatment group / change in tumor volume of the control group) × 100%; c. Statistical analysis (t tests) of the tumor volume of the treatment group and the tumor volume of the PBS control group were performed on the 14th day after group administration. ***P < 0.001.

[0760] Throughout the experiment, the animals were active and ate well during the dosing period. The weight of animals in Group G1 increased to a certain extent, while the weight of animals in Group G2 did not decrease significantly, indicating that the animals tolerated the test product well. The weight changes of all animals are shown in Table 11.

[0761] Table 11. Effects of the test articles on the body weight of mice

[0762] Note: a. Mean ± standard error; b. Body weights of the drug-treated group and the PBS control group were statistically analyzed (ttests) on the 14th day after group administration.

[0763] Example 8 In vivo efficacy of anti-BCMA×GPRC5D×CD3 trispecific antibody in hPBMC-NCG mouse MOLP8 tumor model

[0764] The in vivo antitumor effect of an anti-BCMA×GPRC5D×CD3 trispecific antibody was investigated in the hPBMC-NCG mouse MOLP8 tumor model.

[0765] Severely immunodeficient mice NCG were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; human peripheral blood mononuclear cells (PBMC) were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.

[0766] Human myeloma MOLP8 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. The cells were cultured in an incubator at 37°C and 5% CO2 in RPMI 1640 medium containing 20% ​​inactivated fetal bovine serum.

[0767] On Day 8, 36 severely immunodeficient NCG mice were inoculated with 5×10 6Human peripheral blood mononuclear cells / 0.2 mL RPMI 1640 / mouse, and then on Day 0, 5 × 10 6 MOLP8 cells / 0.2mL RPMI 1640+Matrigel / mouse. When the average tumor volume reached about 160.6mm3, 24 suitable mice were selected for enrollment based on mouse tumor volume and PBMC reconstruction (hCD45% proportion) and randomly assigned to 4 experimental groups, with 6 mice in each group. On days 0, 3, and 7 after grouping, blank control PBS, control antibody Talquetamab, Tri8H5BH563-Fab, and TriBH563CD3G111-1G4S trispecific antibodies (0.3mg / kg) were administered intraperitoneally to the mice, respectively. Tumor volume was monitored by vernier caliper measurement twice a week during the experiment.

[0768] Treatment with the anti-GPRC5D×CD3 control antibody Talquetamab and the anti-BCMA×GPRC5D×CD3 trispecific antibody Tri8H5BH563-Fab resulted in tumor growth inhibition compared to the PBS group, but there was no significant difference, with TGIs of 39% and 46.4%, respectively. Treatment with TriBH563CD3G111-1G4S resulted in significant tumor growth inhibition compared to the PBS group, with TGIs of 46.4% and 73.8%, respectively. The results are shown in Figure 20 and Table 12 below.

[0769] Table 12. Inhibition of MOLP8 tumor growth by the test article

[0770] Note: a. Mean ± standard error; b. TGI = (1 - change in tumor volume in the treatment group / change in tumor volume in the control group) × 100%; c. Statistical analysis (one-way ANOVA) of the tumor volumes in the treatment group and the PBS control group was performed on the 10th day after group administration. *P < 0.05.

[0771] Throughout the experiment, the animals were active and ate well during the dosing period. Animals in groups G1-G3 all showed some weight gain, indicating good tolerance to the test article. Weight changes for all animals are shown in Table 13.

[0772] Table 13. Effects of the test articles on the body weight of mice

[0773] Note: a. Mean ± standard error; b. Body weight of the drug-treated group and the PBS control group were statistically analyzed on the 10th day of administration (One-way ANOVA), *P<0.05.

[0774] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. Any or all features discussed above and throughout this application can be combined in various embodiments of the present invention. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will become apparent from this specification and accompanying drawings and from the appended claims.

[0775] Sequence information

Claims

1. A VHH antibody that specifically binds to BCMA, comprising three complementarity determining regions (CDRs) contained in the VHH represented by any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23, Preferably, the CDR sequences are according to the IMGT definition.

2. The VHH antibody of claim 1, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 9 or 13, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; or (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 5, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 6, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:7; or (iii) VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

7.

3. The VHH antibody of claim 1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NO: 4, 8, 10, 12, 14, 15, 16, 18, 21, 22 or 23, preferably, the amino acid changes do not occur in the CDR region.

4. A heavy chain antibody that specifically binds to BCMA, comprising the VHH antibody according to any one of claims 1 to 3.

5. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1 to 3 connected to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3 or human IgG4, optionally, the VHH antibody is connected to the Fc region via a hinge region or a portion thereof, optionally, the amino acid sequence of the hinge region portion is EPKSS (SEQ ID NO: 94) or EPKSC (SEQ ID NO: 96).

6. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1 to 3 linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1, IgG2, IgG3 or IgG4, optionally comprising a L234A / L235A mutation, a D265A mutation and a P329A mutation, preferably, the Fc region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 80, 90, 97 or 98; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, 90, 97 or 98; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 80, 90, 97 or 98.

7. The heavy chain antibody of claim 4, (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 44-46 and 48-55; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 44-46 and 48-55; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 44 to 46 and 48 to 55, and preferably, the amino acid changes do not occur in the CDR regions.

8. The VHH antibody of any one of claims 1 to 3, or the heavy chain antibody of any one of claims 4 to 7, wherein the antibody is a chimeric antibody or a humanized antibody.

9. A multispecific antibody comprising a first antigen binding region and a second antigen binding region, and optionally a third antigen binding region, wherein the second antigen binding region specifically binds to BCMA, and comprises the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody.

10. The multispecific antibody of claim 9, wherein the first antigen binding region specifically binds CD3.

11. The multispecific antibody of claim 10, wherein the first antigen-binding region comprises a VH and a VL, wherein the VH comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL comprises three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein (i) HCDR1, HCDR2 and HCDR3 are the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:34, and LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:35; or (ii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:28, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:29, HCDR3 consists of the amino acid sequence shown in SEQ ID NO:30, LCDR1 consists of the amino acid sequence shown in SEQ ID NO:31, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:32, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

33.

12. The multispecific antibody of claim 11, wherein the first antigen binding region comprises VH and VL, wherein The VH comprises or consists of the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and / or The VL contains the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

13. The multispecific antibody of any one of claims 10-12, wherein the first antigen binding region is a Fab or scFv that specifically binds to CD3.

14. The multispecific antibody of claim 13, wherein the Fab comprises VH and CH1 of the first antigen binding region, and optionally part of the hinge region (e.g., EPKSS and EPKSC), wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

15. The multispecific antibody of claim 14, wherein the CH1 (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 88; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:

88.

16. The multispecific antibody of claim 15, wherein the Fab heavy chain of the first antigen binding region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 11; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 11; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 11; and / or the Fab light chain of the first antigen binding region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 60; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 60; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:

60.

17. The multispecific antibody of claim 13, wherein the scFv comprises a heavy chain variable region VH comprising a first antigen binding region from the N-terminus to the C-terminus, a linker and a light chain variable region of the first antigen binding region.

18. The multispecific antibody of claim 17, wherein the scFv comprises a sequence as shown in SEQ ID NO: 109 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 109 and having the same CDRs.

19. The multispecific antibody of any one of claims 9-18, wherein the second antigen binding region is the VHH of any one of claims 1-3 and 8.

20. The multispecific antibody of any one of claims 9-19, wherein the multispecific antibody is a trispecific antibody and comprises a third antigen binding region that specifically binds to GPRC5D. The multispecific antibody of claim 20 , wherein the third antigen-binding region comprises or consists of an anti-GPRC5D VHH.

22. The multispecific antibody of claim 21, wherein the anti-GPRC5D VHH comprises or consists of a heavy chain variable region comprising (i) three complementarity determining regions (CDRs) contained in the VH set forth in any one of SEQ ID NO: 27 or 106, or (ii) complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24, VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

26.

23. The multispecific antibody of claim 22, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NO: 27 or 106; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 27 or 106; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 27 or 106, and preferably, the amino acid changes do not occur in the CDR regions.

24. The multispecific antibody of claim 20, wherein the third antigen-binding region comprises a VH and a VL, wherein the VH comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL comprises three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein (i) HCDR1, HCDR2 and HCDR3 are the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:42, and LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:43; or (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 39, and LCD R2 comprises or consists of the amino acid sequence of SEQ ID NO:40, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

41.

25. The multispecific antibody of claim 24, wherein the third antigen binding region comprises VH and VL, wherein The VH comprises or consists of the amino acid sequence of SEQ ID NO:42, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and / or The VL contains the amino acid sequence shown in SEQ ID NO:43 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

26. The multispecific antibody of claim 24 or 25, wherein the third antigen binding region is a Fab that specifically binds to GPRC5D.

27. The multispecific antibody of claim 26, wherein the Fab comprises VH and CH1 of the third antigen binding region, and optionally the CH1 comprises a portion of the hinge region (e.g., EPKSS and EPKSC), wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

28. The multispecific antibody of claim 27, wherein the CH1 (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 88; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:

88.

29. The multispecific antibody of claim 28, wherein the Fab heavy chain of the third antigen binding region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 110; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 110; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 110; and / or the third antigen binding region of the Fab light chain (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 62; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 62; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:

62.

30. The multispecific antibody of any one of claims 9-29, wherein the multispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are identical or different.

31. The multispecific antibody of claim 30, wherein the two Fc regions are different, and preferably, corresponding knob mutations and hole mutations are introduced into the two Fc regions, respectively.

32. The multispecific antibody of claim 31, wherein d) one Fc-region polypeptide comprises the knob mutation T366W and the other Fc-region polypeptide comprises the hole mutations T366S, L368A and Y407V, or e) one Fc-region polypeptide comprises the knob mutations T366W and Y349C and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V and S354C, or f) one Fc-region polypeptide comprises knob mutations T366W and S354C, and the other Fc-region polypeptide comprises hole mutations T366S, L368A, Y407V, and Y349C; Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, for example, one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; for example, a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

33. The bispecific antibody of any one of claims 30-32, wherein one or both of the Fc regions comprise a hinge region, such as EPKSS (SEQ ID NO: 94) or EPKSC (SEQ ID NO: 96).

34. The multispecific antibody of claim 32, wherein (i) Fc region containing knob mutation a) comprises or consists of the amino acid sequence SEQ ID NO: 47, 86 or 87; or b) comprising or consisting of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO:47, 86 or 87 and comprising knob mutations (such as S354C and T366W); and / or (ii) Fc region containing hole mutation a) comprises or consists of the amino acid sequence SEQ ID NO: 84, 85 or 111; or b) comprises or consists of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 84, 85 or 111 and comprising hole mutations (such as Y349C, T366S, L368A and Y407V).

35. The multispecific antibody of any one of claims 9 to 34, which is a bispecific antibody and comprises a first antigen binding region, a second antigen binding region and an Fc dimer, wherein the first antigen binding region is a scFv fragment that specifically binds to CD3, and the second antigen binding region is a VHH that specifically binds to BCMA, such as the VHH of any one of claims 1 to 3 and 8.

36. The multispecific antibody of claim 35, comprising one or two VHHs that specifically bind to BCMA, an scFv of an anti-CD3 antibody, and an Fc heterodimer, wherein The scFv comprises VH-VL (optionally, the VH and VL are fused via a linker), the VHH comprises or consists of a heavy chain variable region, wherein the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the first Fc region (eg, comprising a knob mutation or comprising a hole mutation) to constitute a first heavy chain; The C-terminus of the one anti-BCMA VHH or two tandem anti-BCMA VHHs is fused to a second Fc region (eg, comprising a hole mutation or comprising a knob mutation) to form a second heavy chain (eg, the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region).

37. The multispecific antibody of claim 36, wherein the The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or The second heavy chain comprises the amino acid sequence shown in any one of SEQ ID NO:56, 102, 103 or 104, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

38. The multispecific antibody of any one of claims 9 to 34, which is a trispecific antibody and comprises a first antigen binding region, a second antigen binding region, a third antigen binding region and an Fc dimer, wherein the first antigen binding region is a scFv fragment or a Fab fragment that specifically binds to CD3, the second antigen binding region is a VHH that specifically binds to BCMA, such as the VHH of any one of claims 1 to 3 and 8, and the third antigen binding region is a VHH or Fab fragment that specifically binds to GPRC5D.

39. The multispecific antibody of claim 38, comprising an anti-GPRC5D VHH, an anti-BCMA VHH, an anti-CD3 Fab fragment, and an Fc dimer, wherein The Fab fragment comprises VH-CH1 and VL-CL, the VHH comprises a heavy chain variable region, the trispecific antibody comprises a first heavy chain, a second heavy chain and a light chain, or consists of said chains, wherein The first heavy chain comprises, from N-terminus to C-terminus, anti-GPRC5D VHH, anti-BCMA VHH, and the first Fc region fused via a linker or not; or anti-BCMA VHH, anti-GPRC5D VHH, and the first Fc region fused via a linker or not The second heavy chain comprises, from N-terminus to C-terminus, an anti-CD3 Fab fragment heavy chain and a second Fc region fused via a linker or not; Light chain: Anti-CD3 Fab fragment light chain.

40. The multispecific antibody of claim 39, wherein The first heavy chain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO:58, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; The second heavy chain comprises or consists of an amino acid sequence as shown in any one of SEQ ID NO:59, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or The light chain comprises the amino acid sequence shown in any one of SEQ ID NO:60, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

41. The multispecific antibody of claim 38, comprising an anti-GPRC5D Fab fragment, an anti-BCMA VHH, an anti-CD3 scFv, and an Fc dimer, wherein The Fab fragment comprises VH-CH1 and VL-CL, the VHH comprises a heavy chain variable region, the scFV comprises VH-VL, the trispecific antibody comprises a first heavy chain, a second heavy chain and a light chain, or consists of said chains, wherein The first heavy chain comprises, from N-terminus to C-terminus: an anti-BCMA VHH and a first Fc region fused via or without a linker; The second heavy chain comprises, from N-terminus to C-terminus, a heavy chain of an anti-GPRC5D Fab fragment fused with or without a linker, an anti-CD3 scFv, and a second Fc region; Light chain: Fab fragment light chain of anti-GPRC5D.

42. The multispecific antibody of claim 41, wherein The first heavy chain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO:56, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; The second heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:61, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or The light chain comprises the amino acid sequence shown in SEQ ID NO:62, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

43. A nucleic acid molecule encoding the VHH antibody of any one of claims 1 to 3 and 8, or the heavy chain antibody of any one of claims 4 to 8, or any one chain of the multispecific antibody of any one of claims 9 to 42, or consisting of said nucleic acid sequence.

44. An expression vector comprising the nucleic acid molecule of claim 43.

45. A host cell comprising the nucleic acid molecule of claim 43 or the expression vector of claim 44, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells.

46. ​​A method for preparing the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the multispecific antibody of any one of claims 9-42, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 43 or the expression vector of claim 44 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

47. An immunoconjugate comprising the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the multispecific antibody of any one of claims 9-42.

48. A pharmaceutical composition or medicament or formulation comprising the VHH antibody of any one of claims 1 to 3 and 8, or the heavy chain antibody of any one of claims 4 to 8, or the multi-specific antibody of any one of claims 9 to 42, or the immunoconjugate of claim 47, and optionally a pharmaceutically acceptable excipient.

49. A pharmaceutical combination product comprising the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the multispecific antibody of any one of claims 9-42, or the immunoconjugate of claim 47, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

50. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the bispecific antibody of any one of claims 9-42, or the immunoconjugate of claim 47, or the pharmaceutical composition or medicament or formulation of claim 48; or the pharmaceutical combination product of claim 49.

51. The method of claim 50, wherein the cancer has elevated protein and / or nucleic acid levels (e.g., elevated expression) of BCMA in tumor cells.

52. The method of claim 50 or 51, wherein the cancer is a solid tumor or a hematological tumor, such as a myeloma, such as multiple myeloma such as metastatic multiple myeloma, colon cancer, rectal cancer, or colorectal cancer.

53. The method of any one of claims 50-52, wherein the method further comprises administration in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

54. A method for detecting the presence of BCMA in a biological sample, comprising (i) contacting a biological sample with the VHH antibody of any one of claims 1 to 3 and 8, or the heavy chain antibody of any one of claims 4 to 8, or the multispecific antibody of any one of claims 9 to 42 under conditions that allow them to bind to BCMA, (ii) detecting whether a complex is formed between the antibody or bispecific antibody and BCMA, The formation of the complex indicates the presence of BCMA.

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