Antigen-binding molecule specifically binding to BCMA, GPRC5d, and CD3, and pharmaceutical use thereof

Abstract

An antigen-binding molecule specifically binding to BCMA, GPRC5D, and CD3, and pharmaceutical use thereof.

Description

Antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 and their pharmaceutical applications

[0001] This application claims priority to Chinese patent application CN202411692186.1, filed on November 25, 2024. Technical Field

[0002] This disclosure pertains to the field of biotechnology, and more specifically, to antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 and their pharmaceutical uses. Background Technology

[0003] Multiple myeloma (MM) is a cancer that originates from plasma cells in the bone marrow, typically characterized by the malignant proliferation of plasma cells and the excessive accumulation of monoclonal immunoglobulins. Complications of the disease include anemia, leukopenia, thrombocytopenia, kidney failure, severe pain, bone loss, and hypercalcemia. The progression of MM is similar to that of other cancers; treatment often leads to the acquisition of new genetic alterations and relapse.

[0004] BCMA (tumor necrosis factor receptor superfamily member 17, TNFRSF17) is a type 3 transmembrane protein with an extracellular region rich in cysteine ​​residues. It works in conjunction with TNFR superfamily factors B cell activating factor receptor-B (BAFF-R) and TACI to regulate B cell proliferation and differentiation into plasma cells. These functional receptors support the long-term survival of B cells by binding to ligands BAFF or APRIL. BCMA is only induced to be expressed in the later stages of memory B cell differentiation into plasma cells, and is expressed only on the surface of plasmablasts and differentiated plasma cells; while in memory B cells, BCMA is expressed in the later stages of differentiation into plasma cells. BCMA is not expressed in B cells, CD34+ hematopoietic stem cells, or other normal tissues. Studies in BCMA knockout mice have demonstrated that BCMA plays an important role only in long-term surviving plasma cells and does not regulate B cell homeostasis. Therefore, compared to CD38, which is widely expressed in normal tissues (especially immune cells and organs), BCMA is a highly specific MM antigen.

[0005] GPRC5D, a G protein-coupled orphan receptor and a seven-transmembrane protein, is specifically and highly expressed in patients with multiple myeloma (MM) and is associated with poor prognosis, while it is almost unexpressed in most normal tissues. Furthermore, GPRC5D expression levels are significantly higher in both newly diagnosed MM and relapsed / refractory MM patients than in healthy individuals. Moreover, GPRC5D expression shows no difference across various aberrant cytosis types, whether 1(q)gain or 13(q)del patients. This association between GPRC5D overexpression and cancer suggests the potential of GPRC5D as a superior therapeutic target for cancer.

[0006] Currently, there is no cure for multiple myeloma (MM), and MM patients often relapse after treatment. Therefore, developing a new targeted immunotherapy is of significant clinical importance. Summary of the Invention

[0007] This disclosure provides an antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, and an antigen-binding molecule that specifically binds to BCMA.

[0008] In one aspect, this disclosure provides an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, comprising a first antigen-binding domain that specifically binds BCMA, a second antigen-binding domain that specifically binds CD3, and a third antigen-binding domain that specifically binds GPRC5D.

[0009] Regarding the first antigen-binding domain that specifically binds to BCMA:

[0010] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the first antigen-binding domain that specifically binds BCMA comprises an immunoglobulin single variable domain, the immunoglobulin single variable domain comprising one, two, or three CDR amino acid sequences from SEQ ID NO: 38, 25, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40; or the immunoglobulin single variable domain comprises one, two, or three CDR amino acid sequences from SEQ ID NO: 34 or 16.

[0011] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the sequence of SEQ ID NO: 38, 25, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40; or the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the sequence of SEQ ID NO: 34 or 16.

[0012] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the sequence of SEQ ID NO: 38 or 25.

[0013] In some embodiments, the immunoglobulin single variable domain, as described in any of the preceding embodiments, specifically binds to antigen-binding molecules of BCMA, GPRC5D, and CD3, comprising CDR1, CDR2, and CDR3, which are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the Kabat numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the IMGT numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the Chothia numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the AbM numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the Contact numbering rules. For example, the immunoglobulin single variable domain comprises one, two, or three CDR amino acid sequences from SEQ ID NO: 38, 25, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40. SEQ ID NO: 38, 25, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40 is an immunoglobulin single variable domain sequence. The encoding rules for the CDR amino acid sequences of the immunoglobulin single variable domain sequence can be defined according to the numbering rules of Kabat, IMGT, Chothia, AbM, or Contact.

[0014] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 168; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15; wherein:

[0015] Where X1 is D or E; X2 is D or E.

[0016] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 14, 20, or 21; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0017] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin has a single variable domain CDR1, CDR2, CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 20; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0018] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 21; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0019] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 14; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0020] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the CDR amino acid sequence (CDR1, CDR2, CDR3) of the single variable domain of the immunoglobulin is defined according to the Kabat numbering rules.

[0021] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the immunoglobulin single variable domain is humanized, reverse-mutated, affinity-matured, T-cell epitope-removed, antibody deamidated, and / or antibody isomerized.

[0022] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the single variable domain of the immunoglobulin is obtained by removing / reducing the TCE, which has one or more variations in one or more CDRs.

[0023] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the immunoglobulin's single variable domain comprises the frame region (FR) of the human antibody.

[0024] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises FR1, FR2, FR3 derived from IGHV3-23*04 and FR4 derived from IGHJ4*01, and is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of F27S, T28V, F29S, V37Y, L45R, W47L, A93N, K94A, L4V, S30T, L78V, G44Q, N73S, S74A, and S30T. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein CDR1 of the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 14, 20, or 21, and CDR3 comprises the amino acid sequence of SEQ ID NO: 15, and the FR of the heavy chain variable region is either unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of F27S, T28V, F29S, V37Y, L45R, W47L, A93N, K94A, L4V, S30T, L78V, G44Q, N73S, S74A, and S30T. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the CDR1 of the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 13, the CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 15, and the FR of the heavy chain variable region is substituted with amino acids comprising L4V, F27S, T28V, F29S, S30T, V37Y, G44Q, L45R, W47L, N73S, S74A, L78V, A93N, and K94A.

[0025] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein: the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 169; wherein,

[0026] Where X3 is V or L; X4 is T or R; X5 is V or A; X6 is T or S; X7 is S or A; X8 is Q or G; X9 is G or S; X 10 For D or E; X 11 For D or E; X 12 For S or N; X 13 For A or S; X 14V or L; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0027] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 170; wherein,

[0028] Where X3 is V or L; X4 is T or R; X5 is V or A; X6 is T or S; X7 is S or A; X8 is Q or G; X9 is G or S; X 10 For D or E; X 11 For D or E; X 12 For S or N; X 13 For A or S; X 14 V or L; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0029] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 188; wherein:

[0030] Where X4 is T or R; X5 is V or A; X7 is S or A; X9 is G or S; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0031] In some embodiments, as described in any of the preceding embodiments, the C-terminus of the immunoglobulin's single variable domain is modified to reduce its binding to pre-ADA or ADA, which is, for example, present in serum. Reduced binding to pre-ADA or ADA means that the molecule binds to pre-ADA (or ADA) with reduced affinity or avidity.

[0032] In some embodiments, such as the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in the preceding one, the C-terminal modification comprises C-terminal modifications derived from WO2023093899A1 (included herein by reference in its entirety) and CN202111429892.3, and the patents that have priority to these patents (included herein by reference in its entirety). Other techniques used in this disclosure for C-terminal modification to reduce antibody binding to pre-ADA are also well known in the art, such as WO2012175741A3 (Ablynx) and WO2013024059A3 (GSK).

[0033] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 171; wherein,

[0034] Where X3 is V or L; X4 is T or R; X5 is V or A; X6 is T or S; X7 is S or A; X8 is Q or G; X9 is G or S; X 10 For D or E; X 11 For D or E; X 12 For S or N; X 13 For A or S; X 14 V or L; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0035] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 189; wherein:

[0036] Where X4 is T or R; X5 is V or A; X7 is S or A; X9 is G or S; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0037] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises SEQ ID NO: 38, 25, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0038] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 38, 25, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40.

[0039] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 25, 16, 22, 23, 24, 26, or 27.

[0040] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 38, 34, 35, 36, 37, 39, or 40.

[0041] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 38.

[0042] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the single variable domain of the immunoglobulin is an anti-BCAM nanobody or VHH.

[0043] Regarding the second antigen-binding domain that specifically binds to CD3:

[0044] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the second antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0045] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 58; and / or the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 59 or 60.

[0046] In some embodiments, antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3, as described in the preceding embodiment, wherein:

[0047] The VH contains one, two, or three HCDR amino acid sequences from SEQ ID NO: 58; and the VL contains one, two, or three LCDR amino acid sequences from SEQ ID NO: 59; or

[0048] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 58; and the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 60.

[0049] In some embodiments, antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3, as described in the preceding embodiment, wherein:

[0050] The VH contains HCDR1, HCDR2, and HCDR3 from the sequence SEQ ID NO: 58; and the VL contains LCDR1, LCDR2, and LCDR3 from the sequence SEQ ID NO: 59; or

[0051] The VH contains HCDR1, HCDR2 and HCDR3 in the sequence SEQ ID NO: 58; and the VL contains LCDR1, LCDR2 and LCDR3 in the sequence SEQ ID NO: 60.

[0052] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD3 are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD3 are defined according to the Kabat numbering rule. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD3 are defined according to the IMGT numbering rule. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD3 are defined according to the Chothia numbering rules. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD3 are defined according to the AbM numbering rules. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3 of the second antigen-binding domain specifically binding CD3 are defined according to the Contact numbering rules. For example, the VH of the second antigen-binding domain that specifically binds to CD3 as described in this disclosure comprises one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 58. The sequence SEQ ID NO: 58 is the VH sequence, and the encoding rules for the HCDR amino acid sequences of the VH sequence can be defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering rules. For example, the VL of the second antigen-binding domain that specifically binds to CD3 as described in this disclosure comprises one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 59 or 60.SEQ ID NO: 59 or 60 is a VL sequence, and the encoding rule for the HCDR amino acid sequence of the VL sequence can be defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering rules.

[0053] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0054] The HCDR1 contains, or is composed of, the amino acid sequence of SEQ ID NO: 47; HCDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO: 48; HCDR3 contains, or is composed of, the amino acid sequence of SEQ ID NO: 49; and the LCDR1 contains, or is composed of, the amino acid sequence of SEQ ID NO: 50; LCDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO: 51; LCDR3 contains, or is composed of, the amino acid sequence of SEQ ID NO: 52; or

[0055] The HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 47; the HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 48; the HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 49; and the LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 53; the LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 54; and the LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 55.

[0056] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding claims, wherein: HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 47; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 48; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 49; and LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 50; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 51; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 52.

[0057] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR amino acid sequence (HCDR1, HCDR2, and HCDR3) of the second antigen-binding domain that specifically binds CD3 and the LCDR amino acid sequence (LCDR1, LCDR2, and LCDR3) are defined according to the Kabat numbering rules.

[0058] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, wherein the second antigen-binding domain that specifically binds CD3 is humanized, reverse-mutated, affinity-matured, T-cell epitope-removed / reduced, antibody deamidated, and / or antibody isomerized.

[0059] In some embodiments, such as the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the VH and VL of the second antigen-binding domain that specifically binds CD3 are humanized and both contain the frame region (FR) of the human antibody.

[0060] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the VH is derived from FR1, FR2, FR3 of IGHV3-72*01 and FR4 of IGHJ1*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 29Y and 108T; and / or the VL contains FR1, FR2, FR3 of IGLV7-46*01 and FR4 of IGLJ2*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 36V, 46G, 49G, and 57G. In some embodiments, such as the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the FR of the heavy chain variable region comprises an amino acid substitution selected from 29Y and 108T; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 50 or 53, LCDR2 comprises the amino acid sequence of SEQ ID NO: 51 or 54, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52 or 55, and the FR of the light chain variable region comprises an amino acid substitution selected from 36V, 46G, 49G, and 57G. In some embodiments, such as the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the FR of the heavy chain variable region comprises an amino acid substitution selected from 29Y and 108T; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 50, LCDR2 comprises the amino acid sequence of SEQ ID NO: 51, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52, and the FR of the light chain variable region comprises an amino acid substitution selected from 36V, 46G, 49G, and 57G.

[0061] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the second antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0062] The VH comprises an amino acid sequence of SEQ ID NO: 58 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL comprises an amino acid sequence of SEQ ID NO: 59 having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[0063] The VH contains SEQ ID NO: 58 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL contains SEQ ID NO: 60 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0064] In some embodiments, antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3, as described in the preceding embodiment, wherein:

[0065] The VH contains, or is composed of, the amino acid sequence of SEQ ID NO: 58; and the VL contains, or is composed of, the amino acid sequence of SEQ ID NO: 59; or

[0066] The VH contains or is composed of the amino acid sequence of SEQ ID NO: 58; and the VL contains or is composed of the amino acid sequence of SEQ ID NO: 60.

[0067] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein: the VH comprises or is composed of the amino acid sequence of SEQ ID NO: 58; and the VL comprises or is composed of the amino acid sequence of SEQ ID NO: 59.

[0068] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein: the VH comprises or is composed of the amino acid sequence of SEQ ID NO: 58; and the VL comprises or is composed of the amino acid sequence of SEQ ID NO: 60.

[0069] Regarding the third antigen-binding domain that specifically binds to GPRC5D:

[0070] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the third antigen-binding domain that specifically binds to GPRC5D comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0071] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 79; and / or the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 80.

[0072] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the third antigen-binding domain that specifically binds to GPRC5D comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0073] The VH contains HCDR1, HCDR1, and HCDR3 from the sequence SEQ ID NO: 79; the VL contains LCDR1, LCDR1, and LCDR3 from the sequence SEQ ID NO: 80.

[0074] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 that specifically bind the third antigen-binding domain of GPRC5D are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 that specifically bind the third antigen-binding domain of GPRC5D are defined according to the Kabat numbering rule. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 that specifically bind the third antigen-binding domain of GPRC5D are defined according to the IMGT numbering rule. In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 that specifically bind the third antigen-binding domain of GPRC5D are defined according to the Chothia numbering rules. In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 that specifically bind the third antigen-binding domain of GPRC5D are defined according to the AbM numbering rules. In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 that specifically bind the third antigen-binding domain of GPRC5D are defined according to the Contact numbering rules. For example, the VH described in this disclosure comprises one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 79. The sequence SEQ ID NO: 79 is the VH sequence, and the encoding rules for the HCDR amino acid sequences of the VH sequence can be defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering rules. For example, the VL described in this disclosure comprises one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 80.SEQ ID NO: 80 is a VL sequence, and the encoding rule for the HCDR amino acid sequence of the VL sequence can be defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering rules.

[0075] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the third antigen-binding domain that specifically binds GPRC5D comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0076] The HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 73; the HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 74; the HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 75; and the LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 76; the LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 77; and the LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 78.

[0077] In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR amino acid sequences (HCDR1, HCDR2, and HCDR3) of the VH and the LCDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of the VL are defined according to the Kabat numbering rules.

[0078] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, wherein the third antigen-binding domain of the GPRC5D-specifically binding molecule is humanized, reverse-mutated, affinity-matured, T-cell epitope-removed / reduced, antibody deamidation-reduced, and / or antibody isomerization-reduced.

[0079] In some embodiments, such as the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the VH and VL of the third antigen-binding domain that specifically binds GPRC5D are humanized and both contain the frame region (FR) of the human antibody.

[0080] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the VH comprises FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of Q1E, A24V, V37M, M69W, R71A, T73K, and A93S; and / or the VL comprises FR1, FR2, FR3 derived from IGKV2-30*02 and FR4 derived from IGKJ4*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of M4L, F36L, and Q37L. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the HCDR1 of the VH comprises the amino acid sequence of SEQ ID NO: 73, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 74, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 75, and the FR of the VH is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of Q1E, A24V, V37M, M69W, R71A, T73K, and A93S; and the LCDR1 of the VL comprises the amino acid sequence of SEQ ID NO: 76, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 77, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 78, and the FR of the VL is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of M4L, F36L, and Q37L. In some embodiments, the above-mentioned variable regions and CDRs are defined according to the Kabat numbering rules.

[0081] In some embodiments, antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3, as described in the preceding embodiment, wherein:

[0082] The VH contains an amino acid sequence of SEQ ID NO: 79 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL contains an amino acid sequence of SEQ ID NO: 80 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0083] In some embodiments, antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3, as described in the preceding embodiment, wherein:

[0084] The VH contains or is composed of the amino acid sequence of SEQ ID NO: 79; and the VL contains or is composed of the amino acid sequence of SEQ ID NO: 80.

[0085] Regarding antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3:

[0086] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, comprises: at least one (e.g., 2, 3, 4, 5, 6) first antigen-binding domain that specifically binds BCMA, at least one (e.g., 2, 3, 4, 5, 6) second antigen-binding domain that specifically binds CD3, and at least one (e.g., 2, 3, 4, 5, 6) third antigen-binding domain that specifically binds GPRC5D.

[0087] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises: two first antigen-binding domains that specifically bind BCMA, one second antigen-binding domain that specifically binds CD3, and one third antigen-binding domain that specifically binds GPRC5D.

[0088] In some embodiments, such as the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the first antigen-binding domain that specifically binds BCMA comprises or is a single variable domain of an immunoglobulin.

[0089] In some embodiments, in the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the second antigen-binding domain specifically binding CD3 and the third antigen-binding domain specifically binding GPRC5D are selected from full-length antibodies, Fab, Fab′, F(ab′)2, Fd, Fv, scFv, dsFv, and sdAb, respectively. In some embodiments, in the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, both the second antigen-binding domain specifically binding CD3 and the third antigen-binding domain specifically binding GPRC5D are Fab. In some embodiments, in the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the second antigen-binding domain specifically binding CD3 is Fab. In some embodiments, in the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the third antigen-binding domain specifically binding GPRC5D is Fab.

[0090] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the second antigen-binding domain specifically binding CD3 or the third antigen-binding domain specifically binding GPRC5D comprises a dimeric titin chain and an obscurin chain. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the second antigen-binding domain specifically binding CD3 comprises a dimeric titin chain and an obscurin chain. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the third antigen-binding domain specifically binding GPRC5D comprises a dimeric titin chain and an obscurin chain.

[0091] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 108 to SEQ ID NO: 126, and the obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 127 to SEQ ID NO: 167. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 126. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the obscurin chain comprises the amino acid sequence of SEQ ID NO: 161. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 126 and the obscurin chain comprises the amino acid sequence of SEQ ID NO: 161.

[0092] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a full-length antibody containing two heavy chains and two light chains. In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a full-length antibody containing two heavy chains and two light chains, wherein a constant region CH1 of one heavy chain of the full-length antibody is replaced by a titin chain, and a constant region CL of the light chain that pairs with the constant region CH1 of the heavy chain is replaced by an obscurin chain; or a constant region CH1 of one heavy chain of the full-length antibody is replaced by an obscurin chain, and a constant region CL of the light chain that pairs with the constant region CH1 of the heavy chain is replaced by a titin chain, wherein the titin chain and the obscurin chain pair up to form a dimer.

[0093] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a full-length antibody containing two heavy chains and two light chains, wherein the full-length antibody contains two antigen-binding domains targeting different antigens. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a second antigen-binding domain specifically binding CD3 and a third antigen-binding domain specifically binding GPRC5D. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a first antigen-binding domain specifically binding BCMA and a third antigen-binding domain specifically binding GPRC5D. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a first antigen-binding domain specifically binding BCMA and a second antigen-binding domain specifically binding CD3.

[0094] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binding to BCMA, GPRC5D, and CD3, wherein one heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab specifically binding to CD3. In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binding to BCMA, GPRC5D, and CD3, wherein one heavy chain of the full-length antibody contains VH and an obscurin chain, and the light chain bound to the heavy chain contains VL and a titin chain, or one heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein VH, VL, obscurin, and titin together form a replaced Fab specifically binding to CD3.

[0095] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binding to BCMA, GPRC5D, and CD3, wherein one heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab specifically binding to GPRC5D. In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule specifically binding to BCMA, GPRC5D, and CD3, wherein one heavy chain of the full-length antibody contains VH and an obscurin chain, and the light chain bound to the heavy chain contains VL and a titin chain, or one heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein VH, VL, obscurin, and titin together form a replaced Fab specifically binding to GPRC5D.

[0096] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a full-length antibody containing two heavy chains and two light chains, wherein the full-length antibody comprises a second antigen-binding domain that specifically binds CD3 and a third antigen-binding domain that specifically binds GPRC5D, specifically comprising one of the following structures:

[0097] (i) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1 and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, the Obscurin chain and the Titin chain together form a replaced Fab that specifically binds to GPRC5D;

[0098] (ii) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1 and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein VH, VL, obscurin chain and titin chain together form a replaced Fab that specifically binds to GPRC5D;

[0099] (iii) One heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, the Obscurin chain, and the Titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab that specifically binds to GPRC5D; and

[0100] (iv) One heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein the VH, VL, obscurin chain and titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains VL and a light chain constant region CL, wherein the VH, VL, CH1 and CL together form a Fab that specifically binds to GPRC5D.

[0101] In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, comprises a full-length antibody containing two heavy chains and two light chains, wherein a single variable domain of the immunoglobulin is operatively linked to the N-terminus or C-terminus of the full-length antibody. In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, wherein the single variable domain of the immunoglobulin is operatively linked to the C-terminus of the full-length antibody. In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, wherein the single variable domain of the immunoglobulin is operatively linked to the C-terminus of the light chain of the full-length antibody. In some embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, wherein the N-terminus of the single variable domain of the immunoglobulin is operatively linked to the C-terminus of the light chain of the full-length antibody.

[0102] In some embodiments, an antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, comprises a full-length antibody containing two heavy chains and two light chains, and further comprises two immunoglobulin single variable domains, wherein the N-terminus of one immunoglobulin single variable domain is attached to the C-terminus of the constant region CL of the light chain of the full-length antibody, and the N-terminus of the other immunoglobulin single variable domain is attached to the C-terminus of an obscurin chain or a titin chain on the light chain of the full-length antibody.

[0103] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 comprises a full-length antibody containing two heavy chains and two light chains, the full-length antibody comprising a second antigen-binding domain that specifically binds CD3 and a third antigen-binding domain that specifically binds GPRC5D, and the antigen-binding molecule further comprising two immunoglobulin single variable domains, specifically comprising one of the following structures:

[0104] (i) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1 and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, Obscurin chain and Titin chain together form a replaced Fab that specifically binds to GPRC5D; the N-terminus of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the Titin chain on the light chain, respectively;

[0105] (ii) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein VH, VL, the obscurin chain, and the titin chain together form a replaced Fab that specifically binds to GPRC5D; the N-terminus of each of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the obscurin chain on the light chain, respectively;

[0106] (iii) One heavy chain of the full-length antibody contains a VH and an Obscurin chain, and a light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin chain, and Titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains a VH and a heavy chain constant region CH1, and a light chain bound to the heavy chain contains a VL and a light chain constant region CL, wherein the VH, VL, CH1, and CL together form a Fab that specifically binds to GPRC5D; the N-termini of the two immunoglobulin single variable domains are respectively connected to the C-termini of the light chain constant region CL and the C-termini of the Titin chain on the light chain; and

[0107] (iv) One heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein the VH, VL, obscurin chain, and titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains VL and a light chain constant region CL, wherein the VH, VL, CH1, and CL together form a Fab that specifically binds to GPRC5D; the N-terminus of each of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the obscurin chain on the light chain, respectively.

[0108] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, as shown in Figure 1A, comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds CD3 and a third antigen-binding domain that specifically binds GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL. The light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, Obscurin, and Titin together form a replaced Fab that specifically binds to GPRC5D; the N-terminus of the two immunoglobulin single variable domains are respectively connected to the C-terminus of the light chain constant region CL and the C-terminus of the Titin chain on the light chain.

[0109] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as shown in Figure 1A, comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds CD3 and a third antigen-binding domain that specifically binds GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and... The light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein VH, VL, the obscurin chain, and the titin chain together form a replaced Fab that specifically binds to GPRC5D; the N-terminus of the two immunoglobulin single variable domains are respectively connected to the C-terminus of the light chain constant region CL and the C-terminus of the obscurin chain on the light chain.

[0110] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, as shown in Figure 1A, comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds CD3 and a third antigen-binding domain that specifically binds GPRC5D. One heavy chain of the full-length antibody contains a VH and an obscurin chain, and the light chain bound to the heavy chain contains a VL and a titin chain, wherein the VH, VL, obscurin, and titin chains together form a substituted Fab that specifically binds CD3. The other heavy chain of the full-length antibody contains a VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains a VL and a light chain constant region CL, wherein the VH, VL, CH1, and CL together form a Fab that specifically binds GPRC5D. The N-terminus of each of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the titin chain on the light chain, respectively.

[0111] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, as shown in Figure 1A, comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds CD3 and a third antigen-binding domain that specifically binds GPRC5D. One heavy chain of the full-length antibody contains a VH and a titin chain, and the light chain bound to the heavy chain contains a VL and an obscurin chain, wherein the VH, VL, obscurin chain, and titin chain together form a substituted Fab that specifically binds CD3. The other heavy chain of the full-length antibody contains a VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains a VL and a light chain constant region CL, wherein the VH, VL, CH1, and CL together form a Fab that specifically binds GPRC5D. The N-terminus of each of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the obscurin chain on the light chain, respectively.

[0112] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the single variable domain of the immunoglobulin is an anti-BCMA nanobody or a VHH. In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, wherein the single variable domain of the immunoglobulin is a VHH.

[0113] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, as shown in Figure 1A, comprises: a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d), wherein the structures shown in formulas (a), (b), (c), and (d) are arranged from the N-terminus to the C-terminus.

[0114] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0115] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0116] (c)[CD3-VH]-[CH1]-[Fc2];

[0117] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0118] The BCMA-VHH is a single variable domain of an immunoglobulin that specifically binds to BCMA; the CD3-VH and CD3-VL are VH and VL of the second antigen-binding domain that specifically binds to CD3; the GPRC5D-VH and GPRC5D-VL are VH and VL of the third antigen-binding domain that specifically binds to GPRC5D.

[0119] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0120] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3.

[0121] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, as shown in Figure 1A, comprises: a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d), wherein the structures shown in formulas (a), (b), (c), and (d) are arranged from the N-terminus to the C-terminus.

[0122] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0123] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0124] (c)[CD3-VH]-[CH1]-[Fc2];

[0125] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0126] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0127] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0128] The BCMA-VHH comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and CDR3 comprises the amino acid sequence of SEQ ID NO: 15;

[0129] The CD3-VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49; the CD3-VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 50, LCDR2 comprises the amino acid sequence of SEQ ID NO: 51, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52.

[0130] The GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 73, HCDR2 comprises the amino acid sequence of SEQ ID NO: 74, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 75; the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 76, LCDR2 comprises the amino acid sequence of SEQ ID NO: 77, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 78.

[0131] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, as shown in Figure 1A, comprises: a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d), wherein the structures shown in formulas (a), (b), (c), and (d) are arranged from the N-terminus to the C-terminus.

[0132] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0133] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0134] (c)[CD3-VH]-[CH1]-[Fc2];

[0135] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0136] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0137] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0138] The BCMA-VHH contains the amino acid sequence of SEQ ID NO: 38; the CD3-VH contains the amino acid sequence of SEQ ID NO: 58; the CD3-VL contains the amino acid sequence of SEQ ID NO: 59; the GPRC5D-VH contains the amino acid sequence of SEQ ID NO: 79; and the GPRC5D-VL contains the amino acid sequence of SEQ ID NO: 80.

[0139] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, as shown in Figure 1A, comprises: a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d), wherein the structures shown in formulas (a), (b), (c), and (d) are arranged from the N-terminus to the C-terminus.

[0140] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0141] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0142] (c)[CD3-VH]-[CH1]-[Fc2];

[0143] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0144] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0145] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0146] The BCMA-VHH contains the amino acid sequence of SEQ ID NO: 38; the CD3-VH contains the amino acid sequence of SEQ ID NO: 58; the CD3-VL contains the amino acid sequence of SEQ ID NO: 59; the GPRC5D-VH contains the amino acid sequence of SEQ ID NO: 79; and the GPRC5D-VL contains the amino acid sequence of SEQ ID NO: 80.

[0147] The titin chain contains the amino acid sequence of SEQ ID NO: 126; the obscurin chain contains the amino acid sequence of SEQ ID NO: 161.

[0148] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises a heavy chain constant region CH1 and a light chain constant region CL; wherein the heavy chain constant region CH1 is a heavy chain constant region CH1 of human IgG1, IgG2, IgG3, IgG4, or variants thereof, and the light chain constant region CL is a light chain constant region CL of human kappa or lambda. In some embodiments, the heavy chain constant region CH1 comprises the amino acid sequence of SEQ ID NO: 89, and the light chain constant region CL comprises the amino acid sequences of SEQ ID NO: 90, 187, and 191. In some embodiments, the heavy chain constant region CH1 comprises the amino acid sequence of SEQ ID NO: 89, and the light chain constant region CL comprises the amino acid sequence of SEQ ID NO: 90.

[0149] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region, wherein the Fc region is an IgG Fc region. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region, wherein the Fc region is an IgG1 Fc region.

[0150] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region containing one or more amino acid substitutions that can reduce the binding of the Fc region to the Fcγ receptor.

[0151] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region, which is the human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, numbered according to the EU index.

[0152] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region containing one or more amino acid substitutions capable of eliminating the binding of the Fc region to Protein A.

[0153] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region, which is the human IgG1 Fc region, and the amino acid at position 435 is R and the amino acid at position 436 is F, numbered according to the EU index.

[0154] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the Fc region can prolong the in vivo half-life of the antigen-binding molecule.

[0155] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein each of Fc1 and Fc2 independently comprises one or more amino acid substitutions that reduce homodimerization of the Fc region.

[0156] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other. Fc1 includes a protruding structure according to a mortar and pestle technique, and Fc2 includes a porous structure according to a mortar and pestle technique. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region where the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index. In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an Fc region, wherein Fc1 is C at position 354 and amino acid W at position 366; and Fc2 is C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407, numbered according to the EU index.

[0157] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising a protrusion structure according to a mortise and tenon technique, and the Fc2 comprising a pore structure according to a mortise and tenon technique. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising a protrusion structure according to a mortise and tenon technique, and the Fc2 comprising a pore structure according to a mortise and tenon technique; the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index. In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising a protrusion structure according to a mortar and pestle technique, and the Fc2 comprising a pore structure according to a mortar and pestle technique; the Fc1 has a C at position 354 and an amino acid W at position 366; and the Fc2 has a C at position 349, an amino acid S at position 366, an amino acid A at position 368, and an amino acid V at position 407, numbered according to the EU index.

[0158] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 81; and Fc2 comprises the amino acid sequence of SEQ ID NO: 83.

[0159] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid W at position 366 of Fc1 is present; and the amino acid S at position 366 of Fc2 is present, the amino acid A at position 368 is present, the amino acid V at position 407 is present, the amino acid R at position 435 is present, and the amino acid F at position 436 is present, numbered according to the EU index.

[0160] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 is C at position 354 and amino acid W at position 366; and Fc2 is C at position 349, amino acid S at position 366, amino acid A at position 368, amino acid V at position 407, amino acid R at position 435, and amino acid F at position 436, numbered according to the EU index.

[0161] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid at position 234 of Fc1 is A, the amino acid at position 235 is A, the amino acid at position 354 is C, and the amino acid at position 366 is W; and the amino acid at position 234 of Fc2 is A, the amino acid at position 235 is A, the amino acid at position 349 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, the amino acid at position 407 is V, the amino acid at position 435 is R, and the amino acid at position 436 is F, numbered according to the EU index.

[0162] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 81; and Fc2 comprises the amino acid sequence of SEQ ID NO: 84.

[0163] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments includes an IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid at position 366 of Fc1 is W, the amino acid at position 435 is R, and the amino acid at position 436 is F; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index.

[0164] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 is C at position 354, W at position 366, R at position 435, and F at position 436; and Fc2 is C at position 349, S at position 366, A at position 368, and V at position 407, numbered according to the EU index.

[0165] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid at position 234 is A, the amino acid at position 235 is A, the amino acid at position 354 is C, the amino acid at position 366 is W, the amino acid at position 435 is R, and the amino acid at position 436 is F; and the amino acid at position 234 is A, the amino acid at position 235 is A, the amino acid at position 349 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index.

[0166] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 82; and Fc2 comprises the amino acid sequence of SEQ ID NO: 83.

[0167] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising an F405L mutation and the Fc2 comprising a K409R mutation.

[0168] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 17; and Fc2 comprises the amino acid sequence of SEQ ID NO: 87.

[0169] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising F405L, H435R, Y436F mutations, and the Fc2 comprising a K409R mutation.

[0170] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 86; and Fc2 comprises the amino acid sequence of SEQ ID NO: 87.

[0171] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising an F405L mutation, and the Fc2 comprising a K409R, H435R, Y436F mutation.

[0172] In some embodiments, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 17; and Fc2 comprises the amino acid sequence of SEQ ID NO: 88.

[0173] In some embodiments, the linker is a peptide linker that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments. In some embodiments, the peptide linker may be a flexible peptide containing 1-50 or 3-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS, or GGGG (SEQ ID NO: 172), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and L2 is a bond, G, GG, GGG, or GGGG, and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linker is represented by the following formula: (GS) a(GGS) b (GGGS) c (GGGGS) d (GGGGG) e a, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 174), (EAAAR)3 (SEQ ID NO: 175), (EGGGK)3 (SEQ ID NO: 176), (EGGGR)3 (SEQ ID NO: 177), (DAAAR)3 (SEQ ID NO: 178), (DAAAK)3 (SEQ ID NO: 179), (DGGGR)3 (SEQ ID NO: 180) or (DGGGK)3 (SEQ ID NO: 181); or the peptide linker is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS (SEQ ID NO: 182), GGGGS (SEQ ID NO: 183), (GGGGS)2 (SEQ ID NO: 184), (GGGGS)3 (SEQ ID NO: 185), and (GGGGS)3 (SEQ ID NO: 186). SEQ ID NO: 185; or the peptide linker is GGGGG (SEQ ID NO: 186). In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 18. In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 91.

[0174] In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, have:

[0175] A first chain containing an amino acid sequence identical to SEQ ID NO: 92 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of its sequence identity; a second chain containing an amino acid sequence identical to SEQ ID NO: 93 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of its sequence identity; a third chain containing an amino acid sequence identical to SEQ ID NO: 94 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of its sequence identity; and a fourth chain containing an amino acid sequence identical to SEQ ID NO: 95 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of its sequence identity; or

[0176] A first chain containing an amino acid sequence of SEQ ID NO: 92 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a second chain containing an amino acid sequence of SEQ ID NO: 93 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a third chain containing an amino acid sequence of SEQ ID NO: 94 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and a fourth chain containing an amino acid sequence of SEQ ID NO: 96 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0177] In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, have:

[0178] A first strand containing the amino acid sequence SEQ ID NO: 92, a second strand containing the amino acid sequence SEQ ID NO: 93, a third strand containing the amino acid sequence SEQ ID NO: 94, and a fourth strand containing the amino acid sequence SEQ ID NO: 95; or

[0179] A first strand containing the amino acid sequence SEQ ID NO: 92, a second strand containing the amino acid sequence SEQ ID NO: 93, a third strand containing the amino acid sequence SEQ ID NO: 94, and a fourth strand containing the amino acid sequence SEQ ID NO: 96.

[0180] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments has a first chain containing the amino acid sequence of SEQ ID NO: 92, a second chain containing the amino acid sequence of SEQ ID NO: 93, a third chain containing the amino acid sequence of SEQ ID NO: 94, and a fourth chain containing the amino acid sequence of SEQ ID NO: 95.

[0181] In some embodiments, the antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3, as described in any of the preceding embodiments, is an antibody that specifically binds BCMA, GPRC5D, and CD3. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a trispecific antibody against BCMA, GPRC5D, and CD3.

[0182] In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 provided herein can specifically bind human antigens (including human BCMA, human BCMAR27P mutant antigen, GPRC5D antigen, and CD3 antigen) or their epitopes, and have good cross-binding ability with monkey antigens (including monkey BCMA, monkey GPRC5D, and monkey CD3) or their epitopes.

[0183] In some embodiments, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 provided in this disclosure can effectively bind to cell lines with different antigen expression levels (the expression levels of human BCMA antigen and human GPRC5D antigen on the cell surface are different).

[0184] In some implementations, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 provided in this disclosure exhibit comparable killing power against cell lines expressing only human BCMA antigen and cell lines expressing only GPRC5D antigen. This indicates that the antibody disclosed in this disclosure is effective not only against cells with high BCMA expression but also against cells with low BCMA expression or those that have lost BCMA and only express GPRC5D, suggesting that the antibody disclosed in this disclosure can cover a wider range of MM patients.

[0185] In some implementations, the antigen-binding molecules specifically binding to BCMA, GPRC5D, and CD3 provided in this disclosure exhibit good killing ability against various myeloma cells (BCMA and GPRC5D expression covers high, medium, and low levels). In some implementations, the antibody disclosed in this disclosure shows little difference in killing ability against various myeloma cells (BCMA and GPRC5D expression covers high, medium, and low levels), suggesting more balanced killing of cells with heterogeneous antigen expression in cancer patients.

[0186] In some implementations, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 provided in this disclosure still exhibit good killing effects on tumor cells under different E / T ratio conditions (especially under low E / T ratio conditions).

[0187] In some implementations, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 provided in this disclosure have good killing ability against CHO-K1 cells and 293T cells overexpressing the BCMA R27P mutant antigen, indicating that the anti-GPRC5DxBCMAxCD3 antibody disclosed in this disclosure is more tolerant to antigen targets with BCMA R27P mutation.

[0188] In some implementations, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 provided in this disclosure have a killing effect on target cells that is less affected by sBCMA (referring to soluble B cell maturation antigen) and exhibit strong tolerance to sBCMA.

[0189] In some embodiments, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 provided in this disclosure bind to human BCMA with a KD value of less than 2 nM (e.g., less than 1.5 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM); and bind to monkey BCMA with a KD value of less than 10 nM (e.g., less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM), wherein the KD value is measured by Biacore.

[0190] In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 provided in this disclosure bind to human BCMA antigen on the cell surface with an EC50 value of less than 20 nM (e.g., less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, or less than 9 nM); and bind to monkey BCMA antigen on the cell surface with an EC50 value of less than 20 nM (e.g., less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, or less than 10 nM); said EC50 value is detected by FACS.

[0191] In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 provided in this disclosure bind to human GPRC5D antigen on the cell surface with an EC50 value of less than 20 nM (e.g., less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM); and bind to monkey GPRC5D antigen on the cell surface with an EC50 value of less than 100 nM (e.g., less than 90 nM, less than 20 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM); said EC50 value is detected by FACS.

[0192] In some embodiments, the antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 provided in this disclosure bind to human CD3 antigens on the cell surface with an EC50 value of less than 50 nM (e.g., less than 40 nM, less than 35 nM, less than 30 nM, less than 28 nM, less than 27 nM, less than 20 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM); said EC50 value is detected by FACS.

[0193] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to BCMA, comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises one, two, or three CDR amino acid sequences from SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40.

[0194] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2, or CDR3 in the sequence of SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40.

[0195] In some embodiments, where the antigen-binding molecule specifically binds to BCMA as described in any of the preceding embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the Kabat numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the IMGT numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the Chothia numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the AbM numbering rule. In some embodiments, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the Contact numbering rule. For example, a single variable domain of an immunoglobulin may contain one, two, or three CDR amino acid sequences from SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40. SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40 are heavy chain variable region sequences. The encoding rules for the CDR amino acid sequences of the heavy chain variable region sequences can be defined according to the numbering rules of Kabat, IMGT, Chothia, AbM, or Contact.

[0196] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding claims, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 168; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15; wherein:

[0197] Where X1 is D or E; X2 is D or E.

[0198] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding claims, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 14, 20, or 21; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0199] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding claims, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 20; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0200] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 21; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0201] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding claims, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 13; CDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 14; and CDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0202] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the CDR amino acid sequence (CDR1, CDR2, CDR3) of the single variable domain of the immunoglobulin is defined according to the Kabat numbering rules.

[0203] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the immunoglobulin single variable domain is humanized, reverse-mutated, affinity-matured, T-cell epitope-removed, antibody deamidated, and / or antibody isomerized.

[0204] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the single variable domain of the immunoglobulin is obtained by removing / reducing the TCE, which has one or more variations in one or more CDRs.

[0205] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any of the preceding embodiments, the immunoglobulin's single variable domain comprises the frame region (FR) of the human antibody.

[0206] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises FR1, FR2, FR3 derived from IGHV3-23*04 and FR4 derived from IGHJ4*01, and is either unsubstituted or contains one or more amino acid substitutions selected from the group consisting of F27S, T28V, F29S, V37Y, L45R, W47L, A93N, K94A, L4V, S30T, L78V, G44Q, N73S, S74A, and S30T. In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, wherein HCDR1 of the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, 20 or 21, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, and the FR of the heavy chain variable region is unsubstituted or comprises one or more amino acid substitutions selected from the group consisting of F27S, T28V, F29S, V37Y, L45R, W47L, A93N, K94A, L4V, S30T, L78V, G44Q, N73S, S74A and S30T. In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, wherein HCDR1 of the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, and the FR of the heavy chain variable region is substituted with amino acids comprising L4V, F27S, T28V, F29S, S30T, V37Y, G44Q, L45R, W47L, N73S, S74A, L78V, A93N, and K94A.

[0207] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to BCMA, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 169; wherein,

[0208] Where X3 is V or L; X4 is T or R; X5 is V or A; X6 is T or S; X7 is S or A; X8 is Q or G; X9 is G or S; X 10 For D or E; X 11 For D or E; X 12 For S or N; X 13 For A or S; X 14 V or L; X 15 For K or R; X 16For P or A; X 17 It can be Q or L.

[0209] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to BCMA, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 170; wherein,

[0210] Where X3 is V or L; X4 is T or R; X5 is V or A; X6 is T or S; X7 is S or A; X8 is Q or G; X9 is G or S; X 10 For D or E; X 11 For D or E; X 12 For S or N;

[0211] X 13 For A or S; X 14 V or L; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0212] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 188; wherein:

[0213] Where X4 is T or R; X5 is V or A; X7 is S or A; X9 is G or S; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0214] In some embodiments, as described in any of the preceding embodiments, the C-terminus of the immunoglobulin's single variable domain is modified to reduce its binding to pre-ADA or ADA, which is, for example, present in serum. Reduced binding to pre-ADA or ADA means that the molecule binds to pre-ADA (or ADA) with reduced affinity or reduced avidity.

[0215] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in the preceding one, the C-terminal modification comprises C-terminal modifications derived from WO2023093899A1 (included herein by reference in its entirety) and CN202111429892.3, and the patents that have priority to these patents (included herein by reference in its entirety). Other techniques used in this disclosure for C-terminal modification to reduce antibody binding to pre-ADA are also well known in the art, such as WO2012175741A3 (Ablynx) and WO2013024059A3 (GSK).

[0216] In some embodiments, as described in any of the preceding embodiments, an antigen-binding molecule that specifically binds to BCMA, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 171; wherein,

[0217] Where X3 is V or L; X4 is T or R; X5 is V or A; X6 is T or S; X7 is S or A; X8 is Q or G; X9 is G or S; X 10 For D or E; X 11 For D or E; X 12 For S or N; X 13 For A or S; X 14 V or L; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0218] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 189; wherein:

[0219] Where X4 is T or R; X5 is V or A; X7 is S or A; X9 is G or S; X 15 For K or R; X 16 For P or A; X 17 It can be Q or L.

[0220] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27 or 40, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with it.

[0221] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27 or 40.

[0222] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 25, 16, 22, 23, 24, 26 or 27.

[0223] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, wherein the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 38, 34, 35, 36, 37, 39 or 40.

[0224] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 38 or 34.

[0225] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 25 or 16.

[0226] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 25.

[0227] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the immunoglobulin single variable domain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 16.

[0228] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments comprises or is an antibody or antigen-binding fragment that specifically binds to BCMA or a fragment thereof. In some embodiments, the antibody or antigen-binding fragment in the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment in the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments is, for example, a recombinant antibody or a fragment thereof. In some embodiments, the antibody or antigen-binding fragment in the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody, a monoclonal antibody, or a multispecific antibody (bispecific antibody, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv).

[0229] In some embodiments, such as the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments, the single variable domain of the immunoglobulin is a single-domain antibody, nanobody, or VHH.

[0230] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments includes an Fc region. In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments includes an Fc region, wherein the Fc region is an IgG Fc region.

[0231] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments includes an Fc region, wherein the Fc region is an IgG1 Fc region.

[0232] In some embodiments, as described in any of the preceding embodiments, the Fc region of the antigen-binding molecule that specifically binds to BCMA may cause the antigen-binding molecule to form a dimer. In some embodiments, the Fc region may prolong the in vivo half-life of the antigen-binding molecule. In some embodiments, as described in any of the preceding embodiments, the Fc region of the antigen-binding molecule that specifically binds to BCMA may contain one or more amino acid substitutions that may reduce the binding of the Fc region to the Fcγ receptor.

[0233] In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, wherein the Fc region is the IgG1 Fc region, and amino acids at positions 234 and 235 are A, numbered according to the EU index. In some embodiments, as described in any of the preceding embodiments, the antigen-binding molecule that specifically binds to BCMA, wherein the Fc region is the human IgG1 Fc region, and amino acids at positions 234 and 235 are A, and amino acids at position 405 are L, numbered according to the EU index.

[0234] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments comprises an IgG1 Fc region comprising the amino acid sequence of SEQ ID NO: 85, 6, or 17.

[0235] In some embodiments, the linker is a peptide linker, specifically binding to the antigen-binding molecule of BCMA as described in any of the preceding embodiments. In some embodiments, the peptide linker may be a flexible peptide comprising 1-50 or 3-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS, or GGGG (SEQ ID NO: 172), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG, and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linker is represented by the following formula: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) e a, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 174), (EAAAR)3 (SEQ ID NO: 175), (EGGGK)3 (SEQ ID NO: 176).

[0236] (EGGGR)3 (SEQ ID NO: 177), (DAAAR)3 (SEQ ID NO: 178), (DAAAK)3 (SEQ ID NO: 179), (DGGGR)3 (SEQ ID NO: 180), or (DGGGK)3 (SEQ ID NO: 181); or the peptide linker is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS (SEQ ID NO: 182), GGGGS (SEQ ID NO: 183), (GGGGS)2 (SEQ ID NO: 184), and (GGGGS)3 (SEQ ID NO: 185); or the peptide linker is GGGGG (SEQ ID NO: 186). In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 18. In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 91.

[0237] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments comprises SEQ ID NO: 31, 19, 28, 29, 30, 32 or 33, or an amino acid sequence having at least 80% sequence identity with it.

[0238] In some embodiments, an antigen-binding molecule that specifically binds to BCMA, as described in any of the preceding embodiments, comprises the amino acid sequence of SEQ ID NO: 31, 19, 28, 29, 30, 32, or 33.

[0239] In some embodiments, an antigen-binding molecule that specifically binds to BCMA, as described in any of the preceding embodiments, comprises the amino acid sequence of SEQ ID NO: 31.

[0240] In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments is a multispecific antibody that specifically binds to BCMA. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments is a bispecific antibody that specifically binds to BCMA and CD3. In some embodiments, the antigen-binding molecule that specifically binds to BCMA as described in any of the preceding embodiments is a trispecific antibody that specifically binds to BCMA, GPRC5D, and CD3.

[0241] Bispecific antibodies that specifically bind to BCMA and CD3

[0242] In some embodiments, a bispecific antibody that specifically binds to BCMA and CD3 comprises a single variable immunoglobulin domain as described in any of the preceding embodiments and an antigen-binding domain that specifically binds to CD3.

[0243] In some embodiments, a bispecific antibody that specifically binds to BCMA and CD3 comprises a single variable immunoglobulin domain as described in any of the preceding claims and an antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain specifically binding to CD3 comprises VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3, and VL comprises LCDR1, LCDR2, and LCDR3, and the CDR sequence is selected from the following group:

[0244] The HCDR1 contains the amino acid sequence of SEQ ID NO: 41, the HCDR2 contains the amino acid sequence of SEQ ID NO: 42, the HCDR3 contains the amino acid sequence of SEQ ID NO: 43, the LCDR1 contains the amino acid sequence of SEQ ID NO: 44, the LCDR2 contains the amino acid sequence of SEQ ID NO: 45, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 46.

[0245] The HCDR1 contains the amino acid sequence of SEQ ID NO: 47, the HCDR2 contains the amino acid sequence of SEQ ID NO: 48, the HCDR3 contains the amino acid sequence of SEQ ID NO: 49, and the LCDR1 contains the amino acid sequence of SEQ ID NO: 50, the LCDR2 contains the amino acid sequence of SEQ ID NO: 51, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 52; and

[0246] The HCDR1 contains the amino acid sequence of SEQ ID NO: 47, the HCDR2 contains the amino acid sequence of SEQ ID NO: 48, the HCDR3 contains the amino acid sequence of SEQ ID NO: 49, the LCDR1 contains the amino acid sequence of SEQ ID NO: 53, the LCDR2 contains the amino acid sequence of SEQ ID NO: 54, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 55.

[0247] In some embodiments, the bispecific antibodies that specifically bind BCMA and CD3, as described in any of the preceding embodiments, have HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the bispecific antibodies that specifically bind BCMA and CD3, as described in any of the preceding embodiments, have HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 defined according to the Kabat numbering rule. In some embodiments, the bispecific antibodies that specifically bind BCMA and CD3, as described in any of the preceding embodiments, have HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 defined according to the IMGT numbering rule. In some embodiments, the bispecific antibodies that specifically bind BCMA and CD3, as described in any of the preceding embodiments, have HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 defined according to the Chothia numbering rules. In some embodiments, the bispecific antibodies that specifically bind BCMA and CD3, as described in any of the preceding embodiments, have HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 defined according to the AbM numbering rules. In some embodiments, the bispecific antibodies that specifically bind BCMA and CD3, as described in any of the preceding embodiments, have HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 defined according to the Contact numbering rules.

[0248] In some embodiments, a bispecific antibody that specifically binds to BCMA and CD3, as described in any of the preceding embodiments, wherein the antigen-binding domain that specifically binds to CD3 is murine, chimeric, humanized, or fully human.

[0249] In some embodiments, such as the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments, the VH and VL of the antigen-binding domain that specifically binds CD3 are humanized and both contain the frame region (FR) of the human antibody.

[0250] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments, wherein the VH and VL of the antigen-binding domain that specifically binds CD3 are selected from the group consisting of:

[0251] The VH comprises SEQ ID NO: 56 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL comprises SEQ ID NO: 57 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it;

[0252] The VH comprises an amino acid sequence of SEQ ID NO: 58 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL comprises an amino acid sequence of SEQ ID NO: 59 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and

[0253] The VH contains SEQ ID NO: 58 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL contains SEQ ID NO: 60 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0254] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments, wherein the VH and VL of the antigen-binding domain that specifically binds CD3 are selected from the group consisting of:

[0255] The specific binding VH contains the amino acid sequence of SEQ ID NO: 56, and the VL contains the amino acid sequence of SEQ ID NO: 57;

[0256] The specific binding VH comprises the amino acid sequence of SEQ ID NO: 58, and the VL comprises the amino acid sequence of SEQ ID NO: 59; and

[0257] The specific binding VH contains the amino acid sequence of SEQ ID NO: 58, and the VL contains the amino acid sequence of SEQ ID NO: 60.

[0258] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, comprises a CD3-specific Fab domain, a BCMA-specific immunoglobulin single variable domain, and an Fc region, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, and the CD3-specific Fab domain and the BCMA-specific immunoglobulin single variable domain are operatively linked to the N-terminus of Fc1 and Fc2, respectively.

[0259] In some embodiments, as described in any of the preceding embodiments, the bispecific antibody that specifically binds to BCMA and CD3, as shown in Figure 1B, comprises:

[0260] A first chain with the structure shown in equation (e), a second chain with the structure shown in equation (f), and a third chain with the structure shown in equation (g), wherein the structures shown in equations (e), (f), and (g) are arranged from the N end to the C end:

[0261] (e)[VHH]-[connector]-[Fc1];

[0262] (f)[VH]-[CH1]-[Fc2];

[0263] (g)[VL]-[CL];

[0264] The VHH is a single variable domain of an immunoglobulin that specifically binds to BCMA, and the VH and VL are VH and VL that specifically bind to the antigen-binding domain of CD3.

[0265] The linker is a peptide linker; the first chain and the second chain are linked together, and the third chain is linked together with the second chain to form a bispecific antibody that specifically binds to BCMA and CD3.

[0266] In some embodiments, as described in any of the preceding embodiments, the bispecific antibody that specifically binds to BCMA and CD3, as shown in Figure 1B, comprises:

[0267] A first chain with the structure shown in equation (e), a second chain with the structure shown in equation (f), and a third chain with the structure shown in equation (g), wherein the structures shown in equations (e), (f), and (g) are arranged from the N end to the C end:

[0268] (e)[VHH]-[connector]-[Fc1];

[0269] (f)[VH]-[CH1]-[Fc2];

[0270] (g)[VL]-[CL];

[0271] The linker is a peptide linker; the first chain and the second chain are linked together, and the third chain is linked together with the second chain to form a bispecific antibody that specifically binds to BCMA and CD3;

[0272] The VHH comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and CDR3 comprises the amino acid sequence of SEQ ID NO: 15;

[0273] The VH and VL are selected from the following group:

[0274] The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 41, HCDR2 comprises the amino acid sequence of SEQ ID NO: 42, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 43; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 44, LCDR2 comprises the amino acid sequence of SEQ ID NO: 45, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 46.

[0275] The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 50, LCDR2 comprises the amino acid sequence of SEQ ID NO: 51, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52; and

[0276] The VH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49; the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 53, LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 55.

[0277] In some embodiments, as described in any of the preceding embodiments, the bispecific antibody that specifically binds to BCMA and CD3, as shown in Figure 1B, comprises:

[0278] A first chain with the structure shown in equation (e), a second chain with the structure shown in equation (f), and a third chain with the structure shown in equation (g), wherein the structures shown in equations (e), (f), and (g) are arranged from the N end to the C end:

[0279] (e)[VHH]-[connector]-[Fc1];

[0280] (f)[VH]-[CH1]-[Fc2];

[0281] (g)[VL]-[CL];

[0282] The linker is a peptide linker; the first chain and the second chain are linked together, and the third chain is linked together with the second chain to form a bispecific antibody that specifically binds to BCMA and CD3;

[0283] The VHH contains the amino acid sequence of SEQ ID NO: 16 or 25;

[0284] The VH and VL are selected from the following group:

[0285] The VH contains the amino acid sequence of SEQ ID NO: 56, and the VL contains the amino acid sequence of SEQ ID NO: 57;

[0286] The VH contains the amino acid sequence of SEQ ID NO: 58, and the VL contains the amino acid sequence of SEQ ID NO: 59; and

[0287] The VH contains the amino acid sequence of SEQ ID NO: 58, and the VL contains the amino acid sequence of SEQ ID NO: 60.

[0288] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, comprises a heavy chain constant region CH1 and a light chain constant region CL; wherein the heavy chain constant region CH1 is the heavy chain constant region CH1 of human IgG1, IgG2, IgG3, IgG4, or variants thereof, and the light chain constant region CL is the light chain constant region CL of human kappa or lambda. In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, wherein the heavy chain constant region CH1 comprises the amino acid sequence of SEQ ID NO: 89, and the light chain constant region CL comprises the amino acid sequence of SEQ ID NO: 90 or 187.

[0289] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an Fc region. In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an IgG Fc region. In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an IgG1 Fc region.

[0290] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, includes an Fc region containing one or more amino acid substitutions that can reduce the binding of the Fc region to the Fcγ receptor.

[0291] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an Fc region, which is the human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, numbered according to the EU index.

[0292] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, includes an Fc region containing one or more amino acid substitutions capable of eliminating the binding of the Fc region to Protein A.

[0293] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an Fc region, which is the human IgG1 Fc region, and the amino acid at position 435 is R and the amino acid at position 436 is F, numbered according to the EU index.

[0294] In some embodiments, such as the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments, the Fc region can prolong the in vivo half-life of the antigen-binding molecule.

[0295] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein each of Fc1 and Fc2 independently comprises one or more amino acid substitutions that reduce homodimerization in the Fc region.

[0296] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, comprises an Fc region containing a first subunit Fc1 and a second subunit Fc2 capable of associating with each other. Fc1 includes a protruding structure according to a mortar and pestle technique, and Fc2 includes a porous structure according to a mortar and pestle technique. In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, comprises an Fc region where the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index. In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an Fc region, wherein Fc1 is C at position 354 and amino acid W at position 366; and Fc2 is C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407, numbered according to the EU index.

[0297] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising a protrusion structure according to a mortise and tenon technique, and the Fc2 comprising a pore structure according to a mortise and tenon technique. In some embodiments, the bispecific antibody that specifically binds BCMA and CD3, as described in any of the preceding embodiments, comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising a protrusion structure according to a mortise and tenon technique, and the Fc2 comprising a pore structure according to a mortise and tenon technique; the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index. In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising a protruding structure according to the mortar and pestle technique, and the Fc2 comprising a porous structure according to the mortar and pestle technique; the Fc1 is C at position 354 and amino acid W at position 366; and the Fc2 is C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407, numbered according to the EU index.

[0298] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 81; and Fc2 comprises the amino acid sequence of SEQ ID NO: 83.

[0299] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid W at position 366 of Fc1 is present; and the amino acid S at position 366, A at position 368, V at position 407, R at position 435, and F at position 436 of Fc2 are numbered according to the EU index.

[0300] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 is C at position 354 and amino acid W at position 366; and Fc2 is C at position 349, amino acid S at position 366, amino acid A at position 368, amino acid V at position 407, amino acid R at position 435, and amino acid F at position 436, numbered according to the EU index.

[0301] In some embodiments, the bispecific antibody that specifically binds to BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid at position 234 of Fc1 is A, the amino acid at position 235 is A, the amino acid at position 354 is C, and the amino acid at position 366 is W; and the amino acid at position 234 of Fc2 is A, the amino acid at position 235 is A, the amino acid at position 349 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, the amino acid at position 407 is V, the amino acid at position 435 is R, and the amino acid at position 436 is F, numbered according to the EU index.

[0302] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 81; and Fc2 comprises the amino acid sequence of SEQ ID NO: 84.

[0303] In some embodiments, the bispecific antibody that specifically binds to BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid at position 366 of Fc1 is W, the amino acid at position 435 is R, and the amino acid at position 436 is F; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index.

[0304] In some embodiments, the bispecific antibody that specifically binds to BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region containing a first subunit Fc1 and a second subunit Fc2 capable of associating with each other. The Fc1 is C at position 354, W at position 366, R at position 435, and F at position 436; and the Fc2 is C at position 349, S at position 366, A at position 368, and V at position 407, numbered according to the EU index.

[0305] In some embodiments, the bispecific antibody that specifically binds to BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein the amino acid at position 234 is A, the amino acid at position 235 is A, the amino acid at position 354 is C, the amino acid at position 366 is W, the amino acid at position 435 is R, and the amino acid at position 436 is F; and the amino acid at position 234 is A, the amino acid at position 235 is A, the amino acid at position 349 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index.

[0306] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 82; and Fc2 comprises the amino acid sequence of SEQ ID NO: 83.

[0307] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 contains an F405L mutation and Fc2 contains a K409R mutation.

[0308] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 17; and Fc2 comprises the amino acid sequence of SEQ ID NO: 87.

[0309] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments includes an IgG1 Fc region containing a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 contains F405L, H435R, Y436F mutations and Fc2 contains a K409R mutation.

[0310] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 86; and Fc2 comprises the amino acid sequence of SEQ ID NO: 87.

[0311] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 comprising an F405L mutation, and the Fc2 comprising a K409R, H435R, Y436F mutation.

[0312] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments comprises an IgG1 Fc region, the IgG1 Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 17; and Fc2 comprises the amino acid sequence of SEQ ID NO: 88.

[0313] In some embodiments, the linker of the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments is a peptide linker. In some embodiments, each peptide linker independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS, or GGGG (SEQ ID NO: 172), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG, and the peptide linker is not a bond. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linker is represented by the following formula: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) ea, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 174), (EAAAR)3 (SEQ ID NO: 175), (EGGGK)3 (SEQ ID NO: 176), (EGGGR)3 (SEQ ID NO: 177), (DAAAR)3 (SEQ ID NO: 178), (DAAAK)3 (SEQ ID NO: 179), (DGGGR)3 (SEQ ID NO: 180) or (DGGGK)3 (SEQ ID NO: 181); or the peptide linker is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS (SEQ ID NO: 182), GGGGS (SEQ ID NO: 183), (GGGGS)2 (SEQ ID NO: 184), (GGGGS)3 (SEQ ID NO: 185), and (GGGGS)3 (SEQ ID NO: 186). SEQ ID NO: 185; or the peptide linker is GGGGG (SEQ ID NO: 186). In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 18. In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 91.

[0314] In some embodiments, the bispecific antibody that specifically binds to BCMA and CD3, as described in any of the preceding embodiments, has the following characteristics:

[0315] i) a first chain containing the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a second chain containing the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and a third chain containing the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0316] ii) a first chain containing the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a second chain containing the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and a third chain containing the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0317] iii) A first chain containing the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a second chain containing the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and a third chain containing the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0318] iv) A first chain containing the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a second chain containing the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and a third chain containing the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0319] v) A first chain comprising the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; a second chain comprising the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; and a third chain comprising the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith; or

[0320] vi) A first chain containing the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; a second chain containing the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; and a third chain containing the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0321] The first chain and the second chain bind to each other, and the second chain and the third chain bind to each other to form a bispecific antibody that specifically binds to BCMA and CD3.

[0322] In some embodiments, the bispecific antibody that specifically binds to BCMA and CD3, as described in any of the preceding embodiments, has the following characteristics:

[0323] i) a first strand containing the amino acid sequence of SEQ ID NO: 65, a second strand containing the amino acid sequence of SEQ ID NO: 66, and a third strand containing the amino acid sequence of SEQ ID NO: 67;

[0324] ii) A first strand containing the amino acid sequence of SEQ ID NO: 68, a second strand containing the amino acid sequence of SEQ ID NO: 66, and a third strand containing the amino acid sequence of SEQ ID NO: 67;

[0325] iii) A first strand containing the amino acid sequence of SEQ ID NO: 65, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 71;

[0326] iv) A first strand containing the amino acid sequence of SEQ ID NO: 68, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 71;

[0327] v) A first strand containing the amino acid sequence of SEQ ID NO: 65, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 72; or

[0328] vi) A first strand containing the amino acid sequence of SEQ ID NO: 68, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 72;

[0329] The first chain and the second chain bind to each other, and the second chain and the third chain bind to each other to form a bispecific antibody that specifically binds to BCMA and CD3.

[0330] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments has a first chain containing the amino acid sequence of SEQ ID NO: 65, a second chain containing the amino acid sequence of SEQ ID NO: 66, and a third chain containing the amino acid sequence of SEQ ID NO: 67; the first chain and the second chain bind to each other, and the second chain and the third chain bind to each other to form a bispecific antibody that specifically binds BCMA and CD3.

[0331] In some embodiments, the bispecific antibody that specifically binds BCMA and CD3 as described in any of the preceding embodiments has a first chain containing the amino acid sequence of SEQ ID NO: 68, a second chain containing the amino acid sequence of SEQ ID NO: 66, and a third chain containing the amino acid sequence of SEQ ID NO: 67; the first chain and the second chain bind to each other, and the second chain and the third chain bind to each other to form a bispecific antibody that specifically binds BCMA and CD3.

[0332] In some embodiments, this disclosure also provides an isolated anti-BCMA antigen-binding molecule that competitively binds to human BCMA antigens (including human BCMA R27P mutant antigens) or their epitopes with the anti-BCMA antigen-binding molecule as described in any of the preceding claims.

[0333] In some embodiments, the anti-BCMA antigen-binding molecules (including bispecific antibodies that specifically bind BCMA and CD3) provided in this disclosure can specifically bind human BCMA antigen, human BCMA R27P mutant antigen, and have good cross-binding ability with monkey BCMA antigen.

[0334] In some embodiments, the anti-BCMA antigen-binding molecules (including bispecific antibodies that specifically bind BCMA and CD3) provided in this disclosure exhibit good killing ability against cell lines expressing human BCMA antigen and / or human BCMA R27P mutant antigen. This indicates that the anti-BCMA antigen-binding molecules disclosed in this disclosure are more resistant to antigen targets containing BCMA R27P mutations.

[0335] In some embodiments, the anti-BCMA antigen-binding molecules (including bispecific antibodies that specifically bind BCMA and CD3) provided in this disclosure bind to human BCMA antigen on the cell surface with an EC50 value of less than 20 nM (e.g., less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, less than 10 nM, or less than 9 nM); and bind to monkey BCMA antigen on the cell surface with an EC50 value of less than 20 nM (e.g., less than 19 nM, less than 18 nM, less than 17 nM, less than 16 nM, less than 15 nM, less than 14 nM, less than 13 nM, less than 12 nM, less than 11 nM, or less than 10 nM); said EC50 value is detected by FACS.

[0336] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to CD3, comprising an antigen-binding domain that specifically binds to CD3, said antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0337] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 58; and / or the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 59 or 60.

[0338] In some embodiments, an antigen-binding molecule that specifically binds to CD3, as described in any of the preceding embodiments, wherein:

[0339] The VH contains one, two, or three HCDR amino acid sequences from SEQ ID NO: 58; and the VL contains one, two, or three LCDR amino acid sequences from SEQ ID NO: 59; or

[0340] The VH contains one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 58; and the VL contains one, two, or three LCDR amino acid sequences from the sequence SEQ ID NO: 60.

[0341] In some embodiments, an antigen-binding molecule that specifically binds to CD3, as described in any of the preceding embodiments, wherein:

[0342] The VH contains HCDR1, HCDR2, and HCDR3 from the sequence SEQ ID NO: 58; and the VL contains LCDR1, LCDR2, and LCDR3 from the sequence SEQ ID NO: 59; or

[0343] The VH contains HCDR1, HCDR2 and HCDR3 in the sequence SEQ ID NO: 58; and the VL contains LCDR1, LCDR2 and LCDR3 in the sequence SEQ ID NO: 60.

[0344] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the CD3-specific antigen-binding domain are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the CD3-specific antigen-binding domain are defined according to the Kabat numbering rule. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the CD3-specific antigen-binding domain are defined according to the IMGT numbering rule. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the CD3-specific antigen-binding domain are defined according to the Chothia numbering rules. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the CD3-specific antigen-binding domain are defined according to the AbM numbering rules. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the CD3-specific antigen-binding domain are defined according to the Contact numbering rules. For example, the VH of the CD3-specific antigen-binding domain described in this disclosure comprises one, two, or three HCDR amino acid sequences from the sequence in SEQ ID NO: 58. The sequence SEQ ID NO: 58 is a VH sequence, and the encoding rule for the HCDR amino acid sequence of the VH sequence can be defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering rules. For example, the VL of the antigen-binding domain that specifically binds to CD3 described in this disclosure includes one, two, or three HCDR amino acid sequences from the sequence SEQ ID NO: 59 or 60. The sequence SEQ ID NO: 59 or 60 is a VL sequence, and the encoding rule for the HCDR amino acid sequence of the VL sequence can be defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering rules.

[0345] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the CD3-specific antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0346] The HCDR1 contains, or is composed of, the amino acid sequence of SEQ ID NO: 47; HCDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO: 48; HCDR3 contains, or is composed of, the amino acid sequence of SEQ ID NO: 49; and the LCDR1 contains, or is composed of, the amino acid sequence of SEQ ID NO: 50; LCDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO: 51; LCDR3 contains, or is composed of, the amino acid sequence of SEQ ID NO: 52; or

[0347] The HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 47; the HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 48; the HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 49; and the LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO: 53; the LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 54; and the LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO: 55.

[0348] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding claims, wherein: HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 47; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 48; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 49; and LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO: 50; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 51; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO: 52.

[0349] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, wherein: the HCDR amino acid sequences (HCDR1, HCDR2, and HCDR3) and LCDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of the antigen-binding domain that specifically binds to CD3 are defined according to the Kabat numbering rules.

[0350] In some embodiments, an antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, wherein: the antigen-binding domain that specifically binds to CD3 is humanized, reverse-mutated, affinity-matured, T-cell epitope-removed / reduced, antibody deamidated, and / or antibody isomerized.

[0351] In some embodiments, such as the CD3-specific antigen-binding molecule described in any of the preceding embodiments, the VH and VL of the CD3-specific antigen-binding domain are humanized and both contain the frame region (FR) of a human antibody.

[0352] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, wherein the VH is derived from FR1, FR2, FR3 of IGHV3-72*01 and FR4 of IGHJ1*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 29Y and 108T; and / or the VL contains FR1, FR2, FR3 of IGLV7-46*01 and FR4 of IGLJ2*01, and is unsubstituted or contains one or more amino acid substitutions selected from the group consisting of 36V, 46G, 49G and 57G. In some embodiments, such as the CD3-specific antigen-binding molecule described in any of the preceding embodiments, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the FR of the heavy chain variable region comprises an amino acid substitution selected from 29Y and 108T; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 50 or 53, LCDR2 comprises the amino acid sequence of SEQ ID NO: 51 or 54, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52 or 55, and the FR of the light chain variable region comprises an amino acid substitution selected from 36V, 46G, 49G, and 57G. In some embodiments, such as the CD3-specific antigen-binding molecule described in any of the preceding embodiments, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49, and the FR of the heavy chain variable region comprises an amino acid substitution selected from 29Y and 108T; and LCDR1 comprises the amino acid sequence of SEQ ID NO: 50, LCDR2 comprises the amino acid sequence of SEQ ID NO: 51, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52, and the FR of the light chain variable region comprises an amino acid substitution selected from 36V, 46G, 49G, and 57G.

[0353] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the CD3-specific antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0354] The VH comprises an amino acid sequence of SEQ ID NO: 58 or having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL comprises an amino acid sequence of SEQ ID NO: 59 having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it; or

[0355] The VH contains SEQ ID NO: 58 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it, and the VL contains SEQ ID NO: 60 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity with it.

[0356] In some embodiments, an antigen-binding molecule that specifically binds to CD3, as described in any of the preceding embodiments, wherein:

[0357] The VH contains, or is composed of, the amino acid sequence of SEQ ID NO: 58; and the VL contains, or is composed of, the amino acid sequence of SEQ ID NO: 59; or

[0358] The VH contains or is composed of the amino acid sequence of SEQ ID NO: 58; and the VL contains or is composed of the amino acid sequence of SEQ ID NO: 60.

[0359] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding claims, wherein: the VH comprises or is composed of the amino acid sequence of SEQ ID NO: 58; and the VL comprises or is composed of the amino acid sequence of SEQ ID NO: 59.

[0360] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding claims, wherein: the VH comprises or is composed of the amino acid sequence of SEQ ID NO: 58; and the VL comprises or is composed of the amino acid sequence of SEQ ID NO: 60.

[0361] In some embodiments, the antigen-binding molecule that specifically binds to CD3, as described in any of the preceding embodiments, comprises or is an antibody or antigen-binding fragment that specifically binds to CD3 or a fragment thereof.

[0362] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, the antibody or antigen-binding fragment is, for example, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, the antibody or antigen-binding fragment is, for example, a recombinant antibody or a fragment thereof. In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, the antibody or antigen-binding fragment is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, a monoclonal antibody, or a multispecific antibody (bispecific antibody, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv). In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, the antigen-binding fragment is Fab, Fab′, F(ab')2, Fd, Fv, scFv, dsFv, or dAb. In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, the antibody is a monoclonal antibody.

[0363] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding claims comprises a heavy chain constant region CH1 and a light chain constant region CL; the heavy chain constant region CH1 is a heavy chain constant region CH1 of human IgG1, IgG2, IgG3, IgG4, or variants thereof, and the light chain constant region CL is a light chain constant region CL of human kappa or lambda. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding claims, the heavy chain constant region CH1 comprises the amino acid sequence of SEQ ID NO: 89, and the light chain constant region CL comprises the amino acid sequence of SEQ ID NO: 90, 187, or 191.

[0364] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments includes an Fc region. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments includes an IgG Fc region. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments includes an IgG1 Fc region.

[0365] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments includes an Fc region comprising one or more amino acid substitutions capable of reducing the binding of the Fc region to the Fcγ receptor. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments includes an Fc region that is the human IgG1 Fc region, and amino acids at positions 234 and 235 are A, numbered according to the EU index.

[0366] In some embodiments, the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments includes an Fc region, said Fc region being the human IgG1 Fc region, and contains an amino acid substitution of F405L or K409R.

[0367] In some embodiments, such as the antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, the Fc region can prolong the in vivo half-life of the antigen-binding molecule.

[0368] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 85, 6, 87, 17, or 190. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 85 or 6. In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding embodiments, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 190.

[0369] In some embodiments, the CD3-specific antigen-binding molecule as described in any of the preceding claims, wherein: the CD3-specific antigen-binding molecule comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 193, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 192, or 173, or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity therewith.

[0370] In some embodiments, an antigen-binding molecule that specifically binds to CD3, as described in any of the preceding embodiments, wherein:

[0371] a. The heavy chain comprises the amino acid sequence of SEQ ID NO: 193, and the light chain comprises the amino acid sequence of SEQ ID NO: 192; or

[0372] b. The heavy chain comprises the amino acid sequence of SEQ ID NO: 193, and the light chain comprises the amino acid sequence of SEQ ID NO: 173.

[0373] In some embodiments, an antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, wherein: the heavy chain comprises the amino acid sequence of SEQ ID NO: 193, and the light chain comprises the amino acid sequence of SEQ ID NO: 192.

[0374] In some embodiments, an antigen-binding molecule that specifically binds to CD3 as described in any of the preceding embodiments, wherein: the heavy chain comprises the amino acid sequence of SEQ ID NO: 193, and the light chain comprises the amino acid sequence of SEQ ID NO: 173.

[0375] In some embodiments, this disclosure also provides an isolated, CD3-specific antigen-binding molecule that competitively binds to human CD3 or its epitopes with a CD3-specific antigen-binding molecule as described in any of the preceding embodiments.

[0376] In some embodiments, the CD3-specific antigen-binding molecules provided in this disclosure are in quantities greater than 9 nM (e.g., greater than 10 nM, greater than 20 nM, greater than 30 nM, greater than 40 nM, greater than 50 nM, greater than 60 nM, greater than 70 nM, greater than 80 nM, greater than 90 nM, greater than 100 nM, greater than 150 nM, greater than 200 nM, greater than 250 nM, greater than 300 nM, greater than 350 nM, greater than 400 nM, greater than 450 nM, greater than 500 nM, greater than 550 nM). KD values ​​(0 nM, greater than 600 nM, greater than 650 nM, greater than 700 nM, greater than 750 nM, greater than 800 nM, greater than 850 nM, greater than 900 nM, greater than 950 nM, greater than 1000 nM, greater than 1100 nM, greater than 1200 nM, greater than 1300 nM, greater than 1400 nM, greater than 1450 nM, or greater than 1500 nM, greater than 1800 nM, or greater than 2000 nM) are bound to human CD3, and the KD values ​​are measured by Biacore.

[0377] In some embodiments, the CD3-specific antigen-binding molecules provided in this disclosure are in quantities less than 2000 nM (e.g., less than 1800 nM, less than 1500 nM, less than 1450 nM, less than 1400 nM, less than 1300 nM, less than 1200 nM, less than 1100 nM, less than 1000 nM, less than 950 nM, less than 900 nM, less than 850 nM, less than 800 nM, less than 750 nM, less than 700 nM, less than 650 nM, less than 600 nM). KD values ​​less than 550 nM, less than 500 nM, less than 450 nM, less than 400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, and less than 9 nM were used to bind to human CD3, and the KD values ​​were measured by Biacore.

[0378] In some implementations, the CD3-specific antigen-binding molecules provided in this disclosure are capable of cross-binding with human and cynomolgus monkey CD3.

[0379] In some embodiments, an antigen-binding molecule that specifically binds to CD3, as described in any of the preceding embodiments, wherein the antigen-binding molecule comprises a first antigen-binding domain that binds to human CD3, and one to four additional antigen-binding domains selected from (a) tumor antigens, or (b) cell surface antigens, receptors, or receptor ligands.

[0380] In another aspect, this disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding claims, or an anti-BCMA antigen-binding molecule as described in any of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0381] In some embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned antigen-binding molecules or antibodies based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned antigen-binding molecules or antibodies. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned antigen-binding molecules or antibodies. In some embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned antigen-binding molecules or antibodies. In some embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned antigen-binding molecules or antibodies. In some embodiments, the pharmaceutical composition contains 0.01%-99.99% of pharmaceutically acceptable carriers, diluents, buffers, or excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% of pharmaceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of pharmaceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% of a pharmaceutically acceptable carrier, diluent, buffer, or excipient. In some embodiments, the pharmaceutical composition contains 2% to 98% of a pharmaceutically acceptable carrier, diluent, buffer, or excipient.

[0382] On the other hand, this disclosure also provides an isolated nucleic acid molecule encoding an antigen-binding molecule that specifically binds to BCMA, GPRC5D and CD3 as described in the preceding claim, and an anti-BCMA antigen-binding molecule as described in the preceding claim.

[0383] On the other hand, this disclosure also provides a carrier containing the nucleic acid molecules described above.

[0384] In another aspect, this disclosure also provides a host cell transformed with the aforementioned vector, the host cell being selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells or insect cells.

[0385] In another aspect, this disclosure also provides methods for producing antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding claims, or anti-BCMA antigen-binding molecules as described in any of the preceding claims, the methods comprising culturing host cells as described in any of the preceding claims in a culture medium to form and accumulate antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding claims, or anti-BCMA antigen-binding molecules as described in any of the preceding claims; and recovering antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding claims, or anti-BCMA antigen-binding molecules as described in any of the preceding claims, from the culture.

[0386] In another aspect, this disclosure also provides a method for in vitro detection or determination of human BCMA, the method comprising using an antigen-binding molecule that specifically binds BCMA, GPRC5D and CD3 as described in any of the preceding claims, or an anti-BCMA antigen-binding molecule as described in any of the preceding claims.

[0387] In another aspect, this disclosure also provides the use of antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding claims, or anti-BCMA antigen-binding molecules as described in any of the preceding claims, in the preparation of reagents for the detection or determination of human BCMA.

[0388] In another aspect, this disclosure also provides a method for in vitro detection or determination of human GPRC5D, the method comprising using an antigen-binding molecule that specifically binds BCMA, GPRC5D and CD3 as described in any of the preceding claims.

[0389] In another aspect, this disclosure also provides the use of antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3 as described in any of the preceding disclosures in the preparation of reagents for the detection or determination of human GPRC5D.

[0390] In another aspect, this disclosure also provides a method for treating a disease or condition, the method comprising administering to a subject a therapeutically effective amount of an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding claims, an anti-BCMA antigen-binding molecule as described in any of the preceding claims, or a pharmaceutical composition as described above. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is hematological cancer. In some embodiments, the cancer is B-cell carcinoma expressing BCMA and / or expressing GPRC5D. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is relapsed or refractory multiple myeloma (RRMM).

[0391] On the other hand, this disclosure also provides the use of antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3 as described in any of the preceding claims, anti-BCMA antigen-binding molecules as described in any of the preceding claims, or pharmaceutical compositions as described above, in the preparation for the treatment of a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is hematological cancer. In some embodiments, the cancer is B-cell carcinoma expressing BCMA and / or expressing GPRC5D. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is relapsed or refractory multiple myeloma (RRMM).

[0392] In another aspect, this disclosure also provides an antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 as described in any of the preceding claims, an anti-BCMA antigen-binding molecule as described in any of the preceding claims, or a pharmaceutical composition as described above, said pharmaceutically, for the treatment of cancer. In some embodiments, the cancer is hematological cancer. In some embodiments, the cancer is B-cell carcinoma expressing BCMA and / or expressing GPRC5D. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is relapsed or refractory multiple myeloma (RRMM). Attached Figure Description

[0393] Figure 1A: The format structure of the anti-GPRC5DxBCMAxCD3 trispecific antibody disclosed herein.

[0394] Figure 1B: The format structure of the anti-BCMA-CD3 bispecific antibody disclosed herein.

[0395] Figure 1C: Format structure of the positive control trispecific antibody JNJ-BGCB491.

[0396] Figure 1D: Format structures of the positive control bispecific antibodies Teclistamab and Talquetamab.

[0397] Figures 2A-2D: Binding activity of the anti-GPRC5DxBCMAxCD3 trispecific antibody to multiple myeloma cell lines in Example 4 of this disclosure. Detailed Implementation

[0398] the term

[0399] The terminology used herein is for descriptive purposes only and is not intended to be limiting. 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 disclosure pertains.

[0400] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0401] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0402] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0403] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0404] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0405] The term "amino acid mutation" encompasses amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In some embodiments, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitutions include substitutions with non-naturally occurring amino acids or with derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used herein to refer to the same amino acid mutation. In this document, the amino acid residue at a specific site may be represented by the format "position + amino acid residue". For example, 102S indicates that the amino acid residue at position 102 is S. C102S indicates that the amino acid residue at position 102 has mutated from C to S. When the residue at a specific site is defined in the claim using the format "position + amino acid residue", the original residue at that site does not limit the scope of protection.

[0406] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, antibody fragments, and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0407] The term "antigen-binding molecule" is used in the broadest sense to encompass molecules that specifically bind antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as well as any molecule containing the aforementioned antibodies, peptides, or antibody fusion proteins, provided they exhibit the desired antigen-binding activity. The antigen-binding molecules described herein comprise a variable region (VH) and a variable region (VL), which together constitute the antigen-binding domain. The antigen-binding molecules described herein comprise a single variable domain of an immunoglobulin (e.g., a single-domain antibody, nanobody, VHH). Exemplarily, the antigen-binding molecules described herein are multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies).

[0408] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH, also known as the variable heavy domain or heavy chain variable region), followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL, also known as the variable light domain or light chain variable domain), followed by a constant light domain (light chain constant region, CL).

[0409] The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this document, referring to antibodies with a structure substantially similar to that of natural antibodies or with a heavy chain containing the Fc region as defined herein. The light chain of a natural intact antibody includes a variable region (VL) and a constant region (CL), with VL located at the amino terminus of the light chain. The constant region includes the κ and λ chains. The heavy chain includes a variable region (VH) and constant regions (CH1, CH2, and CH3), with VH located at the amino terminus of the heavy chain and the constant region located at the carboxyl terminus. CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0410] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies; based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more bispecific antibodies; and based on whether the structure is symmetrical, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. These antibodies have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively large molecular weight. The Fc fragment helps in antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, and 1Fab-IgG. TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb , IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).

[0411] The term "multispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to multiple different antigens or at least two different antigenic epitopes of the same antigen. In this document, "anti-BCMA, GPRC5D, and CD3 antibody" is a multispecific antibody, optionally a trispecific antibody, containing three distinct antigen-binding domains, one of which binds antigen BCMA, one of which binds antigen GPRC5D, and one of which binds CD3. In this document, "anti-BCMA and CD3 antibody" is a multispecific antibody, optionally a bispecific antibody, containing two distinct antigen-binding domains, one of which binds antigen BCMA and one of which binds CD3. The term "multispecific antibody" is used herein in the broadest sense and specifically covers antibodies with multi-epitope specificity. Multispecific antibodies include... However, this includes, but is not limited to, antibodies containing heavy chain variable domains (VH) and light chain variable domains (VL), where VH and VL together constitute an antigen-binding domain (where VH / VL have multi-epitope specificity); antibodies having two or more VH and VL domains (each VH / VL unit binds to a different epitope); antibodies having two or more single variable domains (each single variable domain binds to a different epitope); full-length antibodies; antibodies containing one or more antibody fragments; and antibodies containing antibody fragments linked covalently or non-covalently. Multispecific antibodies can be bispecific antibodies, trispecific antibodies, biantibodies, or similar molecules (see, for example, PNAS). (Description of bispecific antibodies in USA 90(14), 6444-8(1993)). The bispecific and trispecific antibodies described herein can bind to any suitable target other than BCMA and / or GPRC5D. "Bispecific antibody" in this document should be understood as an antibody having two distinct antigen-binding domains defined by different antibody sequences. "Trispecific antibody" in this document should be understood as an antibody having three distinct antigen-binding modules defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes within a single target.

[0412] The term "variable region" or "variable domain" of an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.

[0413] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0414] Table 1. Relationship between CDR numbering systems

[0415] Unless otherwise stated, the variable regions and CDRs in this disclosure embodiment are governed by the "Kabat" numbering rule. Although the Kabat numbering rule is used in specific implementations to define amino acid residues, corresponding technical solutions using other numbering systems are considered equivalent.

[0416] "Antigen-binding fragments" encompass full-length antibodies, Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibodies (sdAb, such as VH or VL or VHH), single-chain Fab (scFab), single-chain antibodies (such as scFv, sc(Fv)2), biantibodies, linear antibodies, bivalent or trivalent or quadrivalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0417] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd, single-domain antibodies (sdAb, such as VH, VL, or VHH), single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (such as scFv, sc(Fv)2); and multispecific antibodies formed from antibody fragments.

[0418] An "immunoglobulin domain" refers to a globular region of an antibody chain (such as the chain of a conventional tetrapeptide chain antibody or a heavy chain antibody), or a polypeptide that is essentially composed of such globular regions. An immunoglobulin domain is characterized by its ability to maintain the immunoglobulin folding properties of the antibody molecule, consisting of two interlayers of approximately seven antiparallel β-sheet strands arranged in two β-sheets, optionally stabilized by conserved disulfide bonds.

[0419] An immunoglobulin variable domain refers to an immunoglobulin domain essentially composed of four "frame regions," referred to in the art and hereinafter as "frame region 1" or "FR1," "frame region 2" or "FR2," "frame region 3" or "FR3," and "frame region 4" or "FR4," respectively. These frame regions are separated by three "complementarity-determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antigen specificity due to the presence of antigen-binding sites.

[0420] "Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain.

[0421] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domain" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), IgNAR, domains, and (single-domain) antibodies (such as dAbs) that are VH domains or derived from VH domains. TM ) and antibodies that are VL domains or derived from VL domains (such as dAbs) TMImmunoglobulin single variable domains, based on and / or derived from heavy chain variable domains (such as VH or VHH domains), are generally preferred. A specific example of an immunoglobulin single variable domain is the “VHH domain” (or simply “VHH”) as defined below. The antigen-binding site of the term “single-domain antibody” is located on and formed by an immunoglobulin single variable domain. This distinguishes “single-domain antibody” from “conventional” immunoglobulins or fragments thereof (such as Fab, scFv, etc.) (where two immunoglobulin variable domains, particularly two variable domains, interact to form the antigen-binding site). Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the antigen-binding site; that is, a total of six CDRs will participate in the formation of the antigen-binding site. In contrast, the binding site of a single-domain antibody is formed by a single VH, VHH, or VL domain. Therefore, the antigen-binding site of a single variable domain of an immunoglobulin is formed by no more than three CDRs.

[0422] The term "VHH domain," also known as heavy chain single-domain antibody, VHH, V H The H domain, VHH antibody fragment, VHH antibody, and nanobody are variable domains of antigen-binding immunoglobulins called "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH" is used to distinguish the variable domain from the heavy chain variable domain (referred to herein as the "VH domain" or "VH") present in conventional 4-chain antibodies and the light chain variable domain (referred herein as the "VL domain" or "VL") present in conventional 4-chain antibodies. The VHH domain can specifically bind epitopes in the absence of other antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains).

[0423] The VHH domain is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain. In some cases, the VHH is derived from naturally occurring antibodies lacking the light chain found in camels or cartilaginous fish, or from synthetic and non-immunogenic VHHs that can be constructed accordingly. In some cases, the VHH is a natural VHH, such as a VHH derived from camels, or a recombinant protein containing a heavy-chain variable domain. In some embodiments, the VHH is derived from species selected from camels, llamas, vicuñas, guanacos, and cartilaginous fish (e.g., but not limited to sharks). In some embodiments, the VHH is derived from alpacas (e.g., but not limited to Huacaya alpaca or Surialpaca). In this disclosure, the terms VHH domain, VHH, VHH antibody fragment, VHH antibody, and "nanobody" and "single-domain antibody" are used interchangeably and refer to an immunoglobulin single variable domain having a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure and specifically binding to an epitope without the presence of another immunoglobulin variable domain.

[0424] VHHs include, but are not limited to, natural antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or those obtained through phage display technology. The total number of amino acid residues in a VHH will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17June, 2009 and WO94 / 04678.

[0425] As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

[0426] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. An Fc region comprises two "Fc subunits" that bind together to form the Fc region. In this disclosure, an Fc subunit can be a CH2 and CH3 domain on a heavy chain, or it can be a CH2 and CH3 domain containing one or more amino acid mutations (e.g., LALA or YTE mutations). In some embodiments, the Fc region comprises two distinct Fc subunits. In some embodiments, the Fc region comprises two identical Fc subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. The Fc regions of antibodies described in this disclosure include those of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some implementations, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or by omitting both the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the numbering rule for the Fc region is the EU numbering system, also known as the EU index.

[0427] The term "Titin chain" refers to a peptide segment or functional variant thereof of the Titin protein containing the Titin Ig-like 152 domain, which is 78-118 amino acids in length and capable of binding to the Obscurin Ig-like 1 or Obscurin-like Ig-like 1 domain to form a dimerized complex.

[0428] The term "Obscurin chain" refers to a peptide segment or functional variant thereof of 87-117 amino acids containing the Obscurin Ig-like 1 domain on the Obscurin protein, or a peptide segment or functional variant thereof of 78-118 amino acids containing the Obscurin-like Ig-like 1 domain on the Obscurin-like 1 protein, wherein the Obscurin chain is capable of binding to the Titin Ig-like 152 domain to form a dimerized complex. The Titin chain and Obscurin chain disclosed herein can be used to replace CH1 and CL in Fab to form a replaced Fab (Fab-S), which does not affect the binding of the antigen-binding molecule to the antigen.

[0429] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0430] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0431] The terms "human antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​residues or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies generated in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of "human antibody" explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0432] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding molecule and an antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0433] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0434] The term “surface plasmon resonance” refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0435] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0436] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0437] The term "antigen" refers to a molecule or molecular part that can be bound by antigen-binding molecules, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding molecules (e.g., antibodies).

[0438] The term "pre-existing anti-drug antibody" or "pre-ADA" refers to an ADA that is already present in a subject or individual to whom a drug (e.g., a protein or peptide drug, more specifically, an antibody drug) will be administered or given. Pre-existing anti-drug antibodies may be present in a subject or individual being used for the first time in an experiment (i.e., a subject or individual to whom the drug has never been administered before).

[0439] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformational epitopes), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen as a protein). The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0440] The terms "specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. - 7 M or smaller (e.g., about 1×10⁻⁶) -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or an epitope within the antigen. In some embodiments, the KD of the antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to antigens or epitopes within antigens may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, such as the cynomolgus (cyno), chimpanzee (chimp), or common marmoset (marmoset)).

[0441] The term "non-binding" means that the antibody cannot bind to an antigen or its epitope in the manner described above for specific binding. For example, when the antibody binds at approximately 1 × 10⁻⁶... -6 M or a larger equilibrium dissociation constant (KD) binds to the antigen or its epitope.

[0442] The terms "anti-BCMA antibody" and "antigen-binding domain that specifically binds to BCMA" refer to antibodies or antigen-binding domains that can bind to human BCMA or its epitopes with sufficient affinity. For example, the affinity (KD) for binding to human BCMA in the Biacore assay is below 3 nM.

[0443] The terms "antigen-binding molecules that specifically bind BCMA, GPRC5D, and CD3" and "anti-BCMA, GPRC5D, and CD3 trispecific antibodies" refer to antigen-binding molecules or antibodies capable of binding with sufficient affinity to BCMA or its epitopes, GPRC5D or its epitopes, and CD3 or its epitopes. For example, the affinity (KD) for binding to human BCMA in the Biacore assay is below 3 nM, and the affinity (EC50 value) for binding to human GPRC5D in the FACS assay is below 20 nM.

[0444] In some embodiments, the term "first antigen-binding domain specifically binding to BCMA" refers to an antigen-binding domain capable of binding to BCMA with a certain affinity, such that molecules containing this antigen-binding domain can be used as diagnostic and / or therapeutic agents targeting BCMA. For example, the first antigen-binding domain specifically binding to BCMA has the following equilibrium dissociation constant (KD) for binding to human BCMA: < about 3 nM, which is measured by surface plasmon resonance assay. In some embodiments, the term "third antigen-binding domain specifically binding to GPRC5D" refers to an antigen-binding domain capable of binding to GPRC5D with a certain affinity, such that molecules containing this antigen-binding domain can be used as diagnostic and / or therapeutic agents targeting GPRC5D. For example, the second antigen-binding domain specifically binding to CD3 has the following EC50 value (FACS detection) for binding to human GPRC5D: < about 20 nM, which is measured by surface plasmon resonance assay. In this disclosure, antigen-binding domains include “antibody,” “antigen-binding fragment,” and “antibody fragment” as defined herein (e.g., Fab, replaced Fab, scFv, or immunoglobulin single variable domain).

[0445] The term "linker" refers to a connecting unit that links two polypeptide fragments. In this document, linkers appearing in the same structural formula may be the same or different. Linkers may be "peptide linkers," containing one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. Linkers used in this document may be the same or different. When a "-" appears in a structural formula, it indicates that the units on either side are directly connected by a covalent bond.

[0446] The term "peptide linker" can be any suitable peptide chain, as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, a peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS, or GGGG (SEQ ID NO: 172), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG, and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 3-15 amino acid residues. In some embodiments, the peptide linker is represented by the following formula: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) ea, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 174), (EAAAR)3 (SEQ ID NO: 175), (EGGGK)3 (SEQ ID NO: 176), (EGGGR)3 (SEQ ID NO: 177), (DAAAR)3 (SEQ ID NO: 178), (DAAAK)3 (SEQ ID NO: 179), (DGGGR)3 (SEQ ID NO: 180) or (DGGGK)3 (SEQ ID NO: 181); or the peptide linker is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS (SEQ ID NO: 182), GGGGS (SEQ ID NO: 183), (GGGGS)2 (SEQ ID NO: 184), (GGGGS)3 (SEQ ID NO: 185), and (GGGGS)3 (SEQ ID NO: 186). SEQ ID NO: 185; or the peptide linker is GGGGG (SEQ ID NO: 186). In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 18. In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 91.

[0447] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0448] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0449] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0450] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0451] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acid includes nucleic acid molecules contained in cells that typically contain such molecules, but which are located extrachromosomally or at a chromosomal location different from their natural chromosomal location. Isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0452] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0453] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions; wherein, when performing optimal alignment of two sequences, gaps are introduced where necessary to obtain the maximum percentage of sequence identity, and no conserved substitutions are considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0454] The terms “fusion” or “linkage” refer to the direct or covalent connection of components (such as antigen-binding domains and Fc domains) via linkers.

[0455] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0456] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. Kluyveromyces polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.

[0457] As used in this application, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformer” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny will have identical DNA contents. This includes mutant progeny that have the same function or biological activity as the original transformed cells from which they were selected.

[0458] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0459] The term "pharmaceutical composition" means a mixture containing one or more antigen-binding molecules that specifically bind BCMA, GPRC5D and CD3 as described herein, antigen-binding molecules that specifically bind BCMA, and other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0460] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is distinct from the active ingredient and non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0461] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably herein. In some embodiments, the individual or subject is a human being.

[0462] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0463] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject's body. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0464] "Treatment" and "treatment" (and their grammatical variations) refer to clinical interventions that attempt to alter the natural processes of the individual being treated, and can be implemented for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, antibodies disclosed herein are used to delay disease onset or slow disease progression.

[0465] The terms "recurrence," "relapse," and "relapsed" refer to the recovery of cancer or disease after a clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence can be considered a recurrence.

[0466] The terms "refractory" or "resistant" refer to cancers or diseases that do not respond to treatment.

[0467] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state. In some embodiments, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, especially a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. A complete therapeutic or preventative effect may not occur after a single dose is administered, but may occur after a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered once or multiple times. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0468] This disclosure discloses antigen-binding molecules that specifically bind to BAMA, GPRC5D, and CD3.

[0469] This disclosure provides antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3. Compared to antibodies that target only one or two of these targets, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 provided in this disclosure allow for more specific targeting of specific cell subpopulations. They possess numerous advantageous properties, such as antigen-binding activity (including specific binding activity to human antigens and good cross-binding activity with monkey antigens), effective binding to cell lines with different antigen expression levels (i.e., varying levels of human BCMA and human GPRC5D antigen expression on the cell surface), good killing ability against various myeloma cell lines (BCMA and GPRC5D expression covers high, medium, and low levels) (suggesting more balanced cell killing against antigen-expressing heterogeneity in cancer patients), good killing effect against tumor cells under different E / T ratios (especially low E / T ratios), and specific binding to the BCMA R27P mutant antigen with good killing ability (suggesting good killing ability against BCMA-mutant cells). R27P mutant antigens are more resistant to their targets, their ability to kill target cells is less affected by sBCMA (they have strong resistance to sBCMA), and they exhibit good therapeutic activity, safety, pharmacokinetic properties, and drug-likeness (such as solubility, viscosity, purity, and stability).

[0470] Exemplary antigen-binding molecules that specifically bind to BAMA, GPRC5D, and CD3

[0471] For example, the antigen-binding molecule disclosed herein that specifically binds to BCMA, GPRC5D, and CD3 comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds to CD3 and a third antigen-binding domain that specifically binds to GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL... Together, they form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains a VH and an Obscurin chain, and the light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin, and Titin chains together form a replaced Fab that specifically binds to GPRC5D; the immunoglobulin's single variable domain is VHH, and the N-terminus of the two BCMA-specific VHHs are respectively connected to the C-terminus of the constant region CL of the light chain and the C-terminus of the Titin chain on the light chain; wherein:

[0472] The VHH comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 13, the amino acid sequence of CDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15;

[0473] The second antigen-binding domain that specifically binds to CD3 comprises VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 47, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 48, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 49; and VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 50, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 51, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 52.

[0474] The third antigen-binding domain that specifically binds to GPRC5D comprises VH and VL. VH comprises HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO: 73, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 74, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 75. VL comprises LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO: 76, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 77, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 78.

[0475] For example, the antigen-binding molecule disclosed herein that specifically binds to BCMA, GPRC5D, and CD3 comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds to CD3 and a third antigen-binding domain that specifically binds to GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL... Together, they form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains a VH and an Obscurin chain, and the light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin, and Titin chains together form a replaced Fab that specifically binds to GPRC5D; the immunoglobulin's single variable domain is VHH, and the N-terminus of the two BCMA-specific VHHs are respectively connected to the C-terminus of the constant region CL of the light chain and the C-terminus of the Titin chain on the light chain; wherein:

[0476] The VHH comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 13, the amino acid sequence of CDR2 is shown in SEQ ID NO: 14, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15;

[0477] The second antigen-binding domain that specifically binds to CD3 comprises VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 47, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 48, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 49; and VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 53, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 54, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 55.

[0478] The third antigen-binding domain that specifically binds to GPRC5D comprises VH and VL. VH comprises HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO: 73, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 74, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 75. VL comprises LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is shown in SEQ ID NO: 76, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 77, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 78.

[0479] For example, the antigen-binding molecule disclosed herein that specifically binds to BCMA, GPRC5D, and CD3 comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds to CD3 and a third antigen-binding domain that specifically binds to GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL... Together, they form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains a VH and an Obscurin chain, and the light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin, and Titin chains together form a replaced Fab that specifically binds to GPRC5D; the immunoglobulin's single variable domain is VHH, and the N-terminus of the two BCMA-specific VHHs are respectively connected to the C-terminus of the constant region CL of the light chain and the C-terminus of the Titin chain on the light chain; wherein:

[0480] The amino acid sequence of VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 58; the amino acid sequence of VL that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 59; the amino acid sequence of VH that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 79; and the amino acid sequence of VL that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 80.

[0481] For example, the antigen-binding molecule disclosed herein that specifically binds to BCMA, GPRC5D, and CD3 comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds to CD3 and a third antigen-binding domain that specifically binds to GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL... Together, they form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains a VH and an Obscurin chain, and the light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin, and Titin chains together form a replaced Fab that specifically binds to GPRC5D; the immunoglobulin's single variable domain is VHH, and the N-terminus of the two BCMA-specific VHHs are respectively connected to the C-terminus of the constant region CL of the light chain and the C-terminus of the Titin chain on the light chain; wherein:

[0482] The amino acid sequence of VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 58; the amino acid sequence of VL that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 59; the amino acid sequence of VH that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 79; and the amino acid sequence of VL that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 80.

[0483] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0484] For example, the antigen-binding molecule disclosed herein that specifically binds to BCMA, GPRC5D, and CD3 comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds to CD3 and a third antigen-binding domain that specifically binds to GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL... Together, they form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains a VH and an Obscurin chain, and the light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin, and Titin chains together form a replaced Fab that specifically binds to GPRC5D; the immunoglobulin's single variable domain is VHH, and the N-terminus of the two BCMA-specific VHHs are respectively connected to the C-terminus of the constant region CL of the light chain and the C-terminus of the Titin chain on the light chain; wherein:

[0485] The amino acid sequence of VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 58; the amino acid sequence of VL that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 60; the amino acid sequence of VH that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 79; and the amino acid sequence of VL that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 80.

[0486] For example, the antigen-binding molecule disclosed herein that specifically binds to BCMA, GPRC5D, and CD3 comprises a full-length antibody containing two heavy chains and two light chains. The full-length antibody includes a second antigen-binding domain that specifically binds to CD3 and a third antigen-binding domain that specifically binds to GPRC5D. The antigen-binding molecule also includes two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL... Together, they form a Fab that specifically binds to CD3; another heavy chain of the full-length antibody contains a VH and an Obscurin chain, and the light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin, and Titin chains together form a replaced Fab that specifically binds to GPRC5D; the immunoglobulin's single variable domain is VHH, and the N-terminus of the two BCMA-specific VHHs are respectively connected to the C-terminus of the constant region CL of the light chain and the C-terminus of the Titin chain on the light chain; wherein:

[0487] The amino acid sequence of the VHH is shown in SEQ ID NO: 38; the amino acid sequence of the VH that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 58; the amino acid sequence of the VL of the VH that specifically binds to the second antigen-binding domain of CD3 is shown in SEQ ID NO: 60; the amino acid sequence of the VH that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 79; and the amino acid sequence of the VL that specifically binds to the third antigen-binding domain of GPRC5D is shown in SEQ ID NO: 80.

[0488] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0489] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have a first, second, third, and fourth chain:

[0490] The amino acid sequence of the first chain is shown in SEQ ID NO: 92, the amino acid sequence of the second chain is shown in SEQ ID NO: 93, the amino acid sequence of the third chain is shown in SEQ ID NO: 94, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 95.

[0491] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have a first, second, third, and fourth chain:

[0492] The amino acid sequence of the first chain is shown in SEQ ID NO: 92; the amino acid sequence of the second chain is formed by adding amino acid residue K to the C-terminus of the sequence shown in SEQ ID NO: 93; the amino acid sequence of the third chain is formed by adding amino acid residue K to the C-terminus of the sequence shown in SEQ ID NO: 94; and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 95.

[0493] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have a first, second, third, and fourth chain:

[0494] The amino acid sequence of the first chain is shown in SEQ ID NO: 92, the amino acid sequence of the second chain is shown in SEQ ID NO: 93, the amino acid sequence of the third chain is shown in SEQ ID NO: 94, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 96.

[0495] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have a first, second, third, and fourth chain:

[0496] The amino acid sequence of the first chain is shown in SEQ ID NO: 92; the amino acid sequence of the second chain is formed by adding amino acid residue K to the C-terminus of the sequence shown in SEQ ID NO: 93; the amino acid sequence of the third chain is formed by adding amino acid residue K to the C-terminus of the sequence shown in SEQ ID NO: 94; and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 96.

[0497] Exemplary immunoglobulin single variable domain

[0498] For example, the antigen-binding molecule that specifically binds to BCMA disclosed herein comprises CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO: 13; the amino acid sequence of CDR2 is shown in SEQ ID NO: 14; and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15.

[0499] For example, the antigen-binding molecule that specifically binds to BCMA disclosed herein is an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is VHH, and the amino acid sequence is shown in SEQ ID NO: 25.

[0500] For example, the antigen-binding molecule that specifically binds to BCMA disclosed herein is an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is VHH, and the amino acid sequence is shown in SEQ ID NO: 16.

[0501] For example, the antigen-binding molecule that specifically binds to BCMA disclosed herein is an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is VHH, and the amino acid sequence is shown in SEQ ID NO: 38.

[0502] For example, the antigen-binding molecule that specifically binds to BCMA disclosed herein is an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is VHH, and the amino acid sequence is shown in SEQ ID NO: 34.

[0503] For example, the antigen-binding molecule that specifically binds to BCMA disclosed herein is an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is VHH, and the amino acid sequence is shown in SEQ ID NO: 31.

[0504] Structure of antigen-binding molecules

[0505] The trispecific antibodies disclosed herein are not limited to a specific molecular structure, as long as they possess the desired antigen-binding function. For example, the trispecific antibodies disclosed herein can be trivalent (1+1+1) or tetravalent (1+1+2). The antigen-binding domain in the trispecific antibody can be any antibody or antibody fragment with antigen-binding activity, fused via a peptide linker. The peptide linkers disclosed herein (e.g., linker 1 and linker 2) can be any suitable peptide chain, as long as the antibody can exhibit the desired antigen-binding activity. For example, the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, GGGS, or GGGG (SEQ ID NO: 172), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG, and the peptide linker is not a bond. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linker is represented by the following formula: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) ea, b, c, d, and e are independent integers greater than or equal to 0; or the peptide linker is selected from: (EAAAK)3 (SEQ ID NO: 174), (EAAAR)3 (SEQ ID NO: 175), (EGGGK)3 (SEQ ID NO: 176), (EGGGR)3 (SEQ ID NO: 177), (DAAAR)3 (SEQ ID NO: 178), (DAAAK)3 (SEQ ID NO: 179), (DGGGR)3 (SEQ ID NO: 180) or (DGGGK)3 (SEQ ID NO: 181); or the peptide linker is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS (SEQ ID NO: 182), GGGGS (SEQ ID NO: 183), (GGGGS)2 (SEQ ID NO: 184), (GGGGS)3 (SEQ ID NO: 185), and (GGGGS)3 (SEQ ID NO: 186). SEQ ID NO: 185; or the peptide linker is GGGGG (SEQ ID NO: 186). In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 18. In some embodiments, the amino acid sequence of the linker is as shown in SEQ ID NO: 91.

[0506] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0507] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0508] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0509] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0510] (c)[CD3-VH]-[CH1]-[Fc2];

[0511] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0512] The BCMA-VHH is a single variable domain of an immunoglobulin that specifically binds to BCMA; the CD3-VH and CD3-VL are VH and VL of the second antigen-binding domain that specifically binds to CD3; the GPRC5D-VH and GPRC5D-VL are VH and VL of the third antigen-binding domain that specifically binds to GPRC5D.

[0513] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0514] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3.

[0515] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0516] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0517] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0518] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0519] (c)[CD3-VH]-[CH1]-[Fc2];

[0520] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0521] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0522] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0523] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 59; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0524] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0525] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0526] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0527] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0528] (c)[CD3-VH]-[CH1]-[Fc2];

[0529] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0530] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0531] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0532] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 59; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0533] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0534] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0535] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0536] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0537] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0538] (c)[CD3-VH]-[CH1]-[Fc2];

[0539] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0540] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0541] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 59; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0542] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0543] The amino acid sequence of linker 1 is shown in SEQ ID NO: 91, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 18.

[0544] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0545] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0546] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0547] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0548] (c)[CD3-VH]-[CH1]-[Fc2];

[0549] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0550] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0551] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 59; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0552] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0553] The amino acid sequence of linker 1 is shown in SEQ ID NO: 91, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 18;

[0554] The amino acid sequence of CH1 is shown in SEQ ID NO: 89, and the amino acid sequence of CL is shown in SEQ ID NO: 90.

[0555] The amino acid sequence of Fc1 is shown in SEQ ID NO: 81, and the amino acid sequence of Fc2 is shown in SEQ ID NO: 84.

[0556] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have a first, second, third, and fourth chain:

[0557] The amino acid sequence of the first chain is shown in SEQ ID NO: 92, the amino acid sequence of the second chain is shown in SEQ ID NO: 93, the amino acid sequence of the third chain is shown in SEQ ID NO: 94, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 95.

[0558] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0559] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0560] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0561] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0562] (c)[CD3-VH]-[CH1]-[Fc2];

[0563] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0564] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0565] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0566] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 60; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0567] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0568] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0569] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0570] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0571] (c)[CD3-VH]-[CH1]-[Fc2];

[0572] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0573] The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist;

[0574] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0575] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 60; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0576] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0577] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0578] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0579] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0580] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0581] (c)[CD3-VH]-[CH1]-[Fc2];

[0582] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0583] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0584] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 60; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0585] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0586] The amino acid sequence of linker 1 is shown in SEQ ID NO: 91, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 18.

[0587] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have:

[0588] A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end:

[0589] (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH];

[0590] (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1];

[0591] (c)[CD3-VH]-[CH1]-[Fc2];

[0592] (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH]

[0593] The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3.

[0594] The amino acid sequence of BCMA-VHH is shown in SEQ ID NO: 38; the amino acid sequence of VH is shown in SEQ ID NO: 58; the amino acid sequence of VL is shown in SEQ ID NO: 60; the amino acid sequence of VH is shown in SEQ ID NO: 79; and the amino acid sequence of VH is shown in SEQ ID NO: 80.

[0595] The amino acid sequence of the titin chain is shown in SEQ ID NO: 126; the amino acid sequence of the obscurin chain is shown in SEQ ID NO: 161.

[0596] The amino acid sequence of linker 1 is shown in SEQ ID NO: 91, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 18;

[0597] The amino acid sequence of CH1 is shown in SEQ ID NO: 89, and the amino acid sequence of CL is shown in SEQ ID NO: 90.

[0598] The amino acid sequence of Fc1 is shown in SEQ ID NO: 81, and the amino acid sequence of Fc2 is shown in SEQ ID NO: 84.

[0599] For example, the antigen-binding molecules that specifically bind to BCMA, GPRC5D, and CD3 disclosed herein have a first, second, third, and fourth chain:

[0600] The amino acid sequence of the first chain is shown in SEQ ID NO: 92, the amino acid sequence of the second chain is shown in SEQ ID NO: 93, the amino acid sequence of the third chain is shown in SEQ ID NO: 94, and the amino acid sequence of the fourth chain is shown in SEQ ID NO: 96.

[0601] In some implementations, the antigen-binding molecule provided in this disclosure is an antibody or an antigen-binding fragment.

[0602] In some implementations, the antigen-binding molecule provided in this disclosure is an antibody fragment.

[0603] In some embodiments, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, particularly a Fab fragment and a single-domain antibody. “Fab” is a monovalent fragment composed of VL, VH, CL, and CH1 domains. A “Fab fragment” can be generated by cleavage of an antibody with papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of the two Fab′ fragments to form the F(ab′)2 molecule. “Fab′-SH” is a Fab′ fragment in which the cysteine ​​residues in the constant region have free thiol groups. “F(ab′)2” is a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region.

[0604] In some embodiments, the antibody fragment is a biantibody, triantibody, or tetraantibody. A biantibody is an antibody fragment having two antigen-binding sites. In some embodiments, the antibody fragment contains linked VH and VL domains on the same polypeptide chain (VH-VL). By using excessively short linkers that prevent pairing between two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites, the two antigens of which may be the same or different. In some embodiments, the antibody fragment contains a VH-VL domain and a VHH domain, thereby creating two antigen-binding sites, the two antigens of which may be the same or different.

[0605] In some implementations, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. A single-domain antibody is a single-domain antibody that does not contain a light chain and can specifically bind to an epitope in the absence of other antigen-binding domains. A single-domain antibody is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain.

[0606] In some embodiments, the antigen-binding molecules or antibodies provided herein are chimeric antibodies. In some embodiments, chimeric antibodies comprise a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, chimeric antibodies are "class-switched" antibodies, wherein the class or subclass has been changed from the class or subclass of the parent antibody.

[0607] In some embodiments, the antigen-binding molecules or antibodies provided herein are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies contain one or more variable regions, wherein the CDR or a portion thereof is derived from the non-human antibody, and the FR or a portion thereof is derived from the human antibody. Optionally, humanized antibodies may also contain a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from a non-human antibody (e.g., an antibody providing the CDR sequence).

[0608] Humanized antibodies and their generation methods are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “resurfuacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describes the “guided selection” method for FR shuffling).

[0609] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of the common sequence of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); mature human (somatic mutant) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and the frame regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0610] Variants of antigen-binding molecules

[0611] In some embodiments, amino acid sequence variants of the antibodies provided in this disclosure are included. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

[0612] Replace, insert, and delete variants

[0613] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Substitution mutagenesis sites of interest include CDR and FR. Conserved substitutions are shown in Table 2 under the heading “Preferred Substitutions.” More substantial variations are provided in Table 2 under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0614] Table 2. Substitution of amino acids

[0615] Based on common side-chain characteristics, amino acids can be grouped as follows:

[0616] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0617] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0618] (3) Acidic: Asp, Glu;

[0619] (4) Alkaline: His, Lys, Arg;

[0620] (5) Residues that affect chain orientation: Gly, Pro;

[0621] (6) Aromatic: Trp, Tyr, Phe.

[0622] Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

[0623] One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity). CDRs can be altered (e.g., substituted), for example, to improve antibody affinity. Such alterations can be made to CDR “hotspots,” residues encoded by codons that undergo mutations at a high frequency during somatic maturation, and / or residues that contact the antigen, while testing the binding affinity of the resulting variant VH or VL. In some implementations of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

[0624] In some embodiments, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) can be made to CDRs that do not materially reduce binding affinity. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0625] One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan mutagenesis." In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0626] Amino acid sequence insertions include fusion of the amino and / or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme or a peptide that extends the serum half-life of the antibody.

[0627] Renovation of Fc District

[0628] In one aspect, in the antigen-binding molecule disclosed herein, one of the second antigen-binding domain specifically binding to CD3 and the third antigen-binding domain specifically binding to GPRC5D is a replaced Fab, said replaced Fab comprising a heavy chain variable region, a light chain variable region, a titin chain, and an obscurin chain. In the replaced Fab, the original CH1 and CL of the Fab are replaced by the titin chain and the obscurin chain. Exemplarily, the sequences of the titin chain and the obscurin chain are shown in Tables 3-1 and 3-2.

[0629] Table 3-1. Amino acid sequence of the titin chain

[0630] Table 3-2. Amino acid sequence of the obscurin chain

[0631] Renovation of Fc District

[0632] In one aspect, the Fc region of the antigen-binding molecule disclosed herein contains one or more amino acid substitutions that reduce its binding to an Fc receptor, such as an Fcγ receptor, and reduce or eliminate effector function. Natural IgG Fc regions, specifically IgG1 or IgG4 Fc regions, may cause the antigen-binding molecule disclosed herein to target cells expressing Fc receptors rather than cells expressing antigens. The modified Fc region of this disclosure exhibits reduced binding affinity to Fc receptors and / or reduced effector function. In some embodiments, the modified Fc region exhibits a 50%, 80%, 90%, or 95% or more reduction in binding affinity to Fc receptors compared to the natural Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the modified Fc region also exhibits reduced binding affinity for complement, such as C1q, compared to the natural Fc region. In some embodiments, the modified Fc region does not exhibit reduced binding affinity for neonatal Fc receptors (FcRn) compared to the natural Fc region. In some embodiments, the modified Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell initiation. For the IgG1 Fc region, amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 replace potentially degraded effector functions. In some embodiments, the Fc region is the human IgG1 Fc region, and amino acid residues at positions 234 and 235 are designated A, numbered according to the EU index. For the IgG4 Fc region, amino acid residues at positions 228 replace potentially degraded effector functions.

[0633] In one aspect, the Fc region of the antigen-binding molecule disclosed herein may also contain disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit. To increase the serum half-life of the antigen-binding molecule, mutations of 252Y, 254T, and 256E may be introduced.

[0634] In one aspect, when the antigen-binding molecule disclosed herein comprises different binding modules fused to the two subunits of the Fc region, undesirable homodimerization may occur. To improve yield and purity, it is therefore advantageous to introduce modifications that promote heterodimerization into the Fc region of the antigen-binding molecule disclosed herein. In some embodiments, the Fc region of the disclosed invention comprises modifications according to the knock-in-hole (KIH) technique, which involves introducing a protrusion (knob) at the interface of the first subunit and a hole (hole) at the interface of the second subunit. This allows the protrusion to be positioned within the hole, promoting heterodimer formation and inhibiting homodimer production. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first subunit with a larger side chain (e.g., tyrosine or tryptophan). The hole is created at the interface of the second subunit by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and hole structures are prepared by altering the nucleic acid encoding the polypeptide; optional amino acid substitutions are shown in Table 4.

[0635] Table 4. KIH mutation combinations

[0636] Besides the mortar and pestle technique, other techniques for modifying the CH3 domain of heavy chains to achieve heterodimerization are also known in the art, such as WO1996027011A1, WO1998050431A2, EP1870459A1, WO2007110205A2, WO2009089004A1, WO2010129304A2, WO2011143545A1, WO2012058768A1, WO2013157954A1 and WO2013096291A2.

[0637] In one aspect, when the CH3 domain of one subunit of the Fc region of the antigen-binding molecule disclosed herein contains a mutation that reduces or eliminates the binding of the Fc region to Protein A, such a mutation produces a heterodimer that binds asymmetrically to Protein A, thereby isolating the heterodimer from the homodimer population via a pH gradient. Exemplary amino acid substitutions that reduce the binding of Fc to Protein A include, but are not limited to, the H435R mutation, the Y436F mutation, or a combination of mutations of H435R and Y436F (IgG1 CH3 domain, EU number). Other techniques for modifying the CH3 domain of Fc to achieve reduced binding to Protein A are also known in the art, for example, those disclosed in US11168111B2 and its family of patents.

[0638] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, the composition of a complete antibody may include an antibody population with all K447 residues and / or G446+K447 residues removed. In some embodiments, the composition of a complete antibody may include an antibody population without the removal of K447 residues and / or G446+K447 residues. In some embodiments, the composition of a complete antibody has an antibody population consisting of a mixture of antibodies with and without K447 residues and / or G446+K447 residues.

[0639] Recombination method

[0640] Antibodies can be produced using recombinant methods. These methods involve providing one or more isolated nucleic acids encoding the antibody.

[0641] In the case of natural antibodies, natural antibody fragments, or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required: one for the light chain or a fragment thereof, and one for the heavy chain or a fragment thereof. These nucleic acids encode the amino acid sequence containing the antibody VL and / or the amino acid sequence containing the antibody VH (e.g., the light and / or heavy chains of the antibody). These nucleic acids can be expressed on the same expression vector or on different expression vectors.

[0642] In one embodiment, this disclosure provides isolated nucleic acids encoding antibodies as described above. Such nucleic acids may independently encode any of the foregoing polypeptide chains (e.g., polypeptide chains comprising any CDR, VH, and / or VL, heavy chains, and / or light chains as described in this disclosure). In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In yet another aspect, this disclosure provides host cells comprising such nucleic acids. In one embodiment, a method for preparing an antibody is provided, wherein the method comprises culturing host cells comprising nucleic acids encoding said antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering said antibody from the host cells (or host cell culture medium).

[0643] To generate antibodies through recombinant synthesis, nucleic acids encoding the protein are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of antibodies), or generated through recombinant methods or obtained through chemical synthesis.

[0644] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, antibodies can be separated from bacterial cell paste in soluble fractions and can be further purified.

[0645] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in antibodies with partial or complete human glycosylation patterns. Suitable host cells for expressing (glycosylated) antibodies can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US 7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa NJ (2004), pp. 255-268.

[0646] Measurement

[0647] The antigen-binding molecules or antibodies disclosed herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In one aspect, the activity of the antigen-binding molecules or antibodies disclosed herein can be tested, for example, by known methods such as ELISA, Biacore, FACS, etc.

[0648] Diagnostic and therapeutic compositions

[0649] In some embodiments, the antigen-binding molecules provided in this disclosure can be used to detect the presence of GPRC5D, BCMA, and / or CD3 in biological samples. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some embodiments, the biological sample comprises cells or tissues, such as tumor tissue.

[0650] In one embodiment, an antigen-binding molecule is provided for use in a diagnostic or detection method. In another aspect, a method is provided for detecting the presence of GPRC5D, BCMA, and / or CD3 in a biological sample. In some embodiments, the method includes contacting the biological sample with the antigen-binding molecule under suitable conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods can be in vitro or in vivo. In one embodiment, antigen-binding molecules are used to select subjects suitable for treatment; for example, GPRC5D, BCMA, and / or CD3 are biomarkers used for patient selection.

[0651] Exemplary conditions that can be diagnosed using the antigen-binding molecules disclosed herein include tumors (such as cancer).

[0652] In some embodiments, a labeled antigen-binding molecule is provided. The label includes, but is not limited to, labels or modules for direct detection (such as fluorescent, chromogenic, electronically dense, chemiluminescent, and radioactive labels) and modules for indirect detection (e.g., modules for indirect detection via enzyme reactions or molecular interactions, such as enzymes or ligands).

[0653] In another aspect, pharmaceutical compositions comprising the said antigen-binding molecule are provided, for example, for any of the following treatment methods. In one aspect, the pharmaceutical composition comprises any antigen-binding molecule provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any antigen-binding molecule provided herein and at least one additional therapeutic agent.

[0654] The pharmaceutical compositions of antigen-binding molecules described in this disclosure are prepared by mixing such antigen-binding molecules, having the desired purity, with one or more optional pharmaceutically acceptable carriers, wherein the pharmaceutical composition is in the form of a lyophilized composition or an aqueous solution. Formulations intended for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane.

[0655] Treatment methods and routes of administration

[0656] Any antigen-binding molecule or antibody provided in this disclosure can be used for therapeutic purposes. In another aspect, this disclosure provides the use of antigen-binding molecules or antibodies in the manufacture or preparation of disease medicaments. In some embodiments, the disease is cancer. In some embodiments, the cancer is hematological cancer. In some embodiments, the cancer is B-cell carcinoma expressing BCMA and / or expressing GPRC5D. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is relapsed or refractory multiple myeloma (RRMM). And the medicament is in the form of an effective amount for the above-described diseases. In some embodiments, the effective amount is a unit daily dose or a unit weekly dose. In one such embodiment, the use further includes administering to a subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). The “subject” according to any of the above embodiments can be a human being.

[0657] In another aspect, pharmaceutical compositions comprising the said antibody are provided, for example, for any of the pharmaceutical uses or treatments described above. In some embodiments, the pharmaceutical composition comprises any antigen-binding molecule or antibody provided in this disclosure, and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0658] The antigen-binding molecules or antibodies disclosed herein can be used alone or in combination with other agents for treatment. For example, the antigen-binding molecules or antibodies disclosed herein can be administered co-administered with at least one other therapeutic agent.

[0659] The antigen-binding molecules or antibodies (and any other therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0660] The antigen-binding molecules or antibodies disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the reagent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The antigen-binding molecules or antibodies may be formulated with or without one or more reagents currently used for the prevention or treatment of the stated condition. The effective amount of such other reagents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at about 1 to 99% of the dosage described herein, or at other dosages, and at any route determined empirically / clinically as appropriate.

[0661] For the purpose of treating a disease, the appropriate dosage of the antigen-binding molecule or antibody disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether it is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either once or after a series of treatments.

[0662] Products

[0663] In another aspect of this disclosure, an article of manufacture (such as a medicine box) is provided, comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and / or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is an antibody of this disclosure. The label or package insert indicates that the use of the composition is for the treatment of a selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing the composition, wherein the composition contains the antibody of this disclosure; and (b) a second container containing the composition, wherein the composition contains an additional therapeutic agent. The article of manufacture in embodiments of this disclosure may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including additional buffers, diluents, filters, needles, and syringes.

[0664] Example

[0665] The present disclosure is further described below with reference to examples and test cases, but these examples and test cases are not intended to limit the scope of the disclosure. Experimental methods in the examples and test cases of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0666] Example 1: Construction, Expression and Purification of BCMA Antigen

[0667] Molecular cloning of the antigen was performed using PCR or gene synthesis methods known in the art. The antigen sequence was constructed into a PHR plasmid vector using Polyethylenimine Max (Mw 40,000*)-High Potency Linear PEI (Polysciences, 24765-1) and... I Reduced Serum Medium, no Phenol Red (Gibco, 11058-021) plasmid was transfected into HEK293 cells to obtain antigen protein.

[0668] Antigen protein sequences: Human BCMA (tumor necrosis factor receptor superfamily member 17 TNFRS17, hBCMA, UniProtKB:Q02223). Using the human BCMA sequence as a template, we designed the hBCMA-ECD(Met1-Ala54)-Llama-Fc protein as an immunogenic antigen and the hBCMA-ECD(Met1-Ala54)-hIgG1-Fc protein as a selection antigen, selecting the extracellular region sequence. Based on the cynomolgus monkey BCMA (NCBI XP_001106892.1, cBCMA) sequence as a template, we designed the cBCMA-ECD(Met1-Ala53)-Llama-Fc protein as an immunogenic antigen and the cBCMA-ECD(Met1-Ala53)-hIgG1-Fc protein as a selection antigen, selecting the extracellular region sequence. The fusion of cBCMA-ECD(Met1-Ala53) with human Fc was performed using a linker to increase flexibility and simplify the purification process. The Llama-Fc sequence was derived from NCBI (AAX73259.1), and this region contains 243 amino acids (Glu1-Ser243). The Fc-tagged antigen was affinity purified using ProteinA-MabSelect SuRe (GE, 17-5438-01) and finally transferred to pH 7.4 PBS.

[0669] The antigen protein and the Fc-containing antigen sequence are as follows:

[0670] Human BCMA full sequence (hBCMA):

[0671] Human BCMA-R27P full sequence (hBCMA-R27P)

[0672] The complete BCMA sequence of the cynomolgus monkey (cBCMA):

[0673] Mouse BCMA full sequence (mBCMA)

[0674] >Llama-Fc sequence:

[0675] hIgG1-Fc sequence:

[0676] >hBCMA-ECD(Met1-Ala54)-Llama-Fc:

[0677] >hBCMA(R27P)-ECD(Met1-Ala54)-Llama-Fc:

[0678] >cBCMA-ECD(Met1-Ala53)-Llama-Fc:

[0679] >hBCMA-ECD(Met1-Ala54)-hIgG1-Fc:

[0680] >cBCMA-ECD(Met1-Ala53)-hIgG1-Fc:

[0681] Connector:

[0682] Note: Underlined text indicates BCMA antigen sequence, and italic text indicates linker sequence.

[0683] The CHO-K1 cell line and 293T cell line overexpressing hBCMA, hBCMA-R27P, and cBCMA were used for BCMA screening / detection.

[0684] The specific procedure is as follows: For CHO-K1 stable transgenic lines, use the lipid transfer method according to the instructions (…). The 3000 Transfection Reagent (Life Technology, catalog number L3000-015) transfects plasmids pMD2.G, psPAX2, and pLVX-Hygro-hBCMA and pLVX-Hygro-hBCMA-R27P containing antigen sequences (hBCMA, hBCMA-R27P) into HEK293T cells, or transfects plasmids pVSVg, pCMV dR8.91 and pCDH-EF1-cynoBCMA-T2A-Luci-P2A-copGFP containing antigen sequences (cyno BCMA) into HEK293T cells to prepare pLVX-Hygro-hBCMA, pLVX-Hygrp-hBCMA-R27P, or pCDH-cyno BCMA lentiviruses. Three days after transfection, cell supernatant was collected and cell debris was filtered out using a 0.45 μm filter membrane. After centrifugation at 50,000 g for 3 hours, the supernatant was removed, and the virus was resuspended in F-12K (Ham's F-12K (Kaighn's) medium, Gibco, 21127022) + 10% FBS medium and used to infect CHO-K1 cells. Two days after infection, 400 μg / mL Hygromycin (Hygromycin B solution, Merbau, MA0210-2) was added, and the cells were cultured for 14 days to obtain hBCMA CHO-K1 cell lines, hBCMA-R27P CHO-K1 cell lines, or cyno BCMA CHO-K1 cell lines. For 293T cells, plasmids pMD2.G, psPAX2, and pLVX-Hygro-hBCMA or pLVX-Hygro-hBCMA-R27P containing the antigen sequence (hBCMA or hBCMA-R27P) were transfected into HEK293T cells using lipid transfection. Three days after transfection, 400 μg / mL Hygromycin (hygromycin B solution, Merbau, MA0210-2) was added, and the cells were cultured for 14 days to obtain hBCMA 293T cell lines or hBCMA-R27P 293T cell lines.

[0685] The antigen proteins used for BCMA screening / detection are biotinylated human BCMA-ECD (Biotinylated Human BCMA-ECD, Acrobiosystem, BCA-H82E4-200μg) and hBCMA-ECD-His (Sinobiological, 10620-H08H).

[0686] Example 2: Preparation and screening of anti-BCMA antibodies

[0687] 2.1 Immunity

[0688] The anti-human BCMA nanobody sequence was obtained by immunizing alpacas. The immunizing agents were human BCMA-ECD(Met1-Ala54)-Llama-Fc (SEQ ID NO:7) and cyno BCMA-ECD(Met1-Ala53)-Llama-Fc (SEQ ID NO:9) proteins. The immunization process is as follows:

[0689] Blood was collected on day 0 before immunization and kept as a negative serum control. The first immunization was administered subcutaneously after mixing the human BCMA-ECD (Met1-Ala54)-Llama-Fc (SEQ ID NO:7) antigen with an equal volume of CFA (Solepro, F5881-10ML). On day 21, the second immunization was administered subcutaneously after mixing the cyno BCMA-ECD (Met1-Ala53)-Llama-Fc (SEQ ID NO:9) antigen with an equal volume of IFA (Solepro, F5506-10ML). On day 28, 10mL of blood was collected to test the titer. On day 42, the human BCMA-ECD (Met1-Ala54)-Llama-Fc (SEQ ID NO:7) antigen and the cyno BCMA-ECD (Met1-Ala53)-Llama-Fc (SEQ ID NO:7) antigen were administered subcutaneously. NO:9) antigen was mixed with an equal volume of IFA and injected subcutaneously for the third vaccination; on day 49, 50 mL of peripheral blood was collected to separate lymphocytes, and the titer was measured at the same time; on day 63, human BCMA-ECD(Met1-Ala54)-Llama-Fc(SEQ ID NO:7) antigen was mixed with an equal volume of IFA and injected subcutaneously for the fourth vaccination; on day 70, 50 mL of peripheral blood was collected to separate lymphocytes, and the titer was measured at the same time.

[0690] The titer assay was performed as follows: Human BCMA-ECD(Met1-Ala54)-human-Fc (SEQ ID NO:10) and cyno BCMA-ECD(Met1-Ala53)-hIgG1-Fc (SEQ ID NO:11) antigens were diluted to 2 μg / mL with 0.05 M carbonate buffer (pH 9.6), and 100 μL / well was added for coating overnight at 4°C. The coating solution was discarded, and the cells were washed three times with PBST. 300 μL of 5% skim milk was added to each well, and the cells were blocked at 37°C for 1 h. After washing three times with PBST, 100 μL / well of serum diluent (serial dilution starting at 1:2000) was added, and the cells were incubated at 37°C for 45 min. After washing five times with PBST, Anti-Alpaca IgG (H+L) was added. Goat antibody (HRP) plus (Alpvhhs, 053-405-005, diluted 1:10000 with PBS), 100 μL / well, incubated at 37°C for 45 min; wash the plate 5 times with PBST. Add TMB chromogenic solution, 100 μL / well, incubate at 37°C for 5 min, then add stop solution to terminate the reaction, 50 μL / well, and measure the optical density at 450 nm.

[0691] 2.2 Phage Library Construction and Screening

[0692] Following the third and fourth immunizations, 50 mL of peripheral blood was collected, and PBMCs were separated according to the instructions for use of lymphocyte separation medium. PrimeScript was then used to separate the cells. TM II. Total RNA was extracted from PBMCs using the 1st Strand cDNA Synthesis Kit. The VHH fragment was identified and recovered using nested PCR, ligated into the pComb3XSS vector, transformed to construct a bacterial library, and then analyzed based on OD... 600 A phage library was constructed by adding helper phage M13KO7 (helper phage:bacteria = 20:1). Subsequently, the alpaca immune phage library was panned 2-3 times (Biotinylated Human BCMA-ECD, BCA-H82E4; Human BCMAProtein-ECD, His Tag, 10620-H08H), and the enriched clones were selected for ELISA screening. The specific procedure is as follows: Single clones were picked and placed into 96-well plates, incubated at 37°C for 4 hours, and then overnight at 30°C. Stable cell lines of human BCMA antigen (1 μg / mL), hBCMA CHO-K1, cBCMA CHO-K1, and CHO-K1 (control) were coated onto 96-well plates respectively. The plates were treated at 37°C for 2 hours, followed by the addition of 200 μL of PBS containing 5% skim milk powder, and incubated overnight at 4°C. Phages were added to 96-well plates coated with antigens or cells for binding, incubated with Anti-M13-HRP secondary antibody, and then the OD450nm reading was detected. Data were processed and analyzed, and the sequencing results were sent for sequencing analysis.

[0693] Finally, a positive clone, VHH5, was obtained through screening. It can bind to human BCMA protein, hBCMACHO-K1, and cBCMA CHO-K1. The VHH5 CDR sequence is shown in Table 5.

[0694] Table 5. BCMA Nanobody VHH CDR Sequence

[0695] Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0696] The sequence of the nanobody obtained from alpaca immunization is as follows:

[0697] VHH5:

[0698] Note: In the above sequences, the underlined part is the CDR sequence determined according to the Kabat numbering system, and the non-underlined part is the FR sequence.

[0699] The recombinant VHH5 sequence with the human IgG1 Fc sequence (SEQ ID NO:17, 6 or 85) was ligated using the EPKSS linker (SEQ ID NO:18) to obtain the chimeric antibody CHI-VHH5.

[0700] hIgG1-Fc-2 sequence

[0701] hFc1 sequences (L234A, L235A, F405L):

[0702] >Connector 2:

[0703] For example:

[0704] >CHI-VHH5:

[0705] Note: In the above sequences, single underscores represent CDR sequences determined according to the Kabat numbering system, and italics represent Fc sequences.

[0706] Example 3: Humanization of anti-BCMA antibody

[0707] 3.1 Humanization of anti-BCMA antibodies

[0708] By comparing the genetic sequence of the heavy and light chain variable regions with the IMGT human antibody heavy and light chain variable region germline gene database using MOE software, germline genes with high homology to VHH5 were selected as templates. The CDRs of the VHH5 nanobody were then transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplarily, in the following specific embodiments, the CDR amino acid residues were determined and annotated using the Kabat numbering system.

[0709] The humanized antibody for VHH5 was prepared by using FR1, FR2, and FR3 of IGHV3-23*04 as the VHH framework region template; and FR4 of IGHJ4*01 as the VHH framework region template. Optionally, amino acid residues 4, 27, 28, 29, 30, 37, 44, 45, 47, 52, 61, 73, 74, 78, 93, and / or 94 of the VHH region of the humanized antibody were substituted, as detailed in Table 6.

[0710] Table 6. Human-centered design of VHH5 Note: For example, L4V means that the 4 L's are changed to V according to the Kabat numbering system, and so on.

[0711] Table 7. CDRs of VHH5 humanized antibodies Note: In the table above, CDR sequences are numbered by Kabat.

[0712] The variable region sequence of the VHH5 humanized antibody is as follows:

[0713] >HuVHH5-1:

[0714] >HuVHH5-2:

[0715] >HuVHH5-3:

[0716] >HuVHH5-4:

[0717] >HuVHH5-5:

[0718] >HuVHH5-6:

[0719] Note: In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the underlined part is the CDR sequence determined according to the Kabat numbering system; the ununderlined part is the FR sequence. ...

Claims

1. An antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3, comprising: Specifically binds to the first antigen-binding domain of BCMA, The second antigen-binding domain that specifically binds to CD3, and It specifically binds to the third antigen-binding domain of GPRC5D; The first antigen-binding domain that specifically binds to BCMA comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in the sequence SEQ ID NO: 38, 25, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40; or The immunoglobulin single variable domain includes CDR1, CDR2, and CDR3 in the sequence SEQ ID NO: 34 or 16.

2. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to claim 1, wherein: The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 168 and CDR3 comprises the amino acid sequence of SEQ ID NO:

15.

3. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1 or 2, wherein: The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 14, 20 or 21, and CDR3 comprises the amino acid sequence of SEQ ID NO:

15. Preferably, a-1. The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 14 and CDR3 comprises the amino acid sequence of SEQ ID NO: 15; a-2. The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 20 and CDR3 comprises the amino acid sequence of SEQ ID NO: 15; a-3. The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 21 and CDR3 comprises the amino acid sequence of SEQ ID NO: 15; More preferably, The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 14 and CDR3 comprises the amino acid sequence of SEQ ID NO:

15.

4. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-3, wherein: The second antigen-binding domain that specifically binds to CD3 includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH includes HCDR1, HCDR2 and HCDR3 in the sequence of SEQ ID NO: 58; and the VL includes LCDR1, LCDR2 and LCDR3 in the sequence of SEQ ID NO: 59 or 60.

5. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-4, wherein: The second antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 47, HCDR2 comprises the amino acid sequence of SEQ ID NO: 48, and HCDR3 comprises the amino acid sequence of SEQ ID NO:

49. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 50 or 53, LCDR2 comprises the amino acid sequence of SEQ ID NO: 51 or 54, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 52 or 55. Preferably, The HCDR1 contains the amino acid sequence of SEQ ID NO: 47, the HCDR2 contains the amino acid sequence of SEQ ID NO: 48, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 49; the LCDR1 contains the amino acid sequence of SEQ ID NO: 50, the LCDR2 contains the amino acid sequence of SEQ ID NO: 51, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 52; or The HCDR1 contains the amino acid sequence of SEQ ID NO: 47, the HCDR2 contains the amino acid sequence of SEQ ID NO: 48, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 49; the LCDR1 contains the amino acid sequence of SEQ ID NO: 53, the LCDR2 contains the amino acid sequence of SEQ ID NO: 54, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

55. More preferably, The HCDR1 contains the amino acid sequence of SEQ ID NO: 47, the HCDR2 contains the amino acid sequence of SEQ ID NO: 48, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 49; the LCDR1 contains the amino acid sequence of SEQ ID NO: 50, the LCDR2 contains the amino acid sequence of SEQ ID NO: 51, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

52.

6. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-5, wherein: The third antigen-binding domain that specifically binds to GPRC5D comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 79; and the VL comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO:

80.

7. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-6, wherein: The third antigen-binding domain that specifically binds to GPRC5D comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises HCDR1, HCDR2, and HCDR3. HCDR1 comprises the amino acid sequence of SEQ ID NO: 73, HCDR2 comprises the amino acid sequence of SEQ ID NO: 74, and HCDR3 comprises the amino acid sequence of SEQ ID NO:

75. The VL comprises LCDR1, LCDR2, and LCDR3. LCDR1 comprises the amino acid sequence of SEQ ID NO: 76, LCDR2 comprises the amino acid sequence of SEQ ID NO: 77, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

78.

8. The antigen-binding molecule that specifically binds to BCMA, GPRC5D and CD3 according to any one of claims 1-7, wherein the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 169, 170, 171, 188, or 189.

9. The antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 according to any one of claims 1-7, wherein the immunoglobulin single variable domain comprises SEQ ID NO: 38, 25, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40, or an amino acid sequence having at least 80% sequence identity with it; Preferably, The immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 38, 25, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27 or 40; More preferably, The immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 38, 34, 35, 36, 37, 39 or 40; Most preferably, The immunoglobulin single variable domain contains the amino acid sequence of SEQ ID NO:

38.

10. The antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 according to any one of claims 1-9, wherein the second antigen-binding domain specifically binding CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein, The VH contains an amino acid sequence of SEQ ID NO: 58 or having at least 80% sequence identity with it, and the VL contains an amino acid sequence of SEQ ID NO: 59 or 60 or having at least 80% sequence identity with it; Preferably, The VH contains the amino acid sequence of SEQ ID NO: 58, and the VL contains the amino acid sequence of SEQ ID NO: 59 or 60; More preferably, The VH contains the amino acid sequence of SEQ ID NO: 58, and the VL contains the amino acid sequence of SEQ ID NO:

59.

11. The antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 according to any one of claims 1-10, wherein the third antigen-binding domain that specifically binds GPRC5D comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein, The VH contains an amino acid sequence of SEQ ID NO: 79 or having at least 80% sequence identity with it, and the VL contains an amino acid sequence of SEQ ID NO: 80 or having at least 80% sequence identity with it; Preferably, The VH contains the amino acid sequence of SEQ ID NO: 79, and the VL contains the amino acid sequence of SEQ ID NO:

80.

12. The antigen-binding molecule that specifically binds BCMA, GPRC5D and CD3 according to any one of claims 1-11, wherein the second antigen-binding domain and the third antigen-binding domain are selected from full-length antibodies, Fab, Fab′, F(ab′)2, Fd, Fv, scFv, or dsFv; Preferably, both the second antigen-binding domain and the third antigen-binding domain are Fab domains. More preferably, both the second antigen-binding domain and the third antigen-binding domain are Fab; the single variable domain of the immunoglobulin is an anti-BCMA nanobody or VHH.

13. The antigen-binding molecule that specifically binds to BCMA, GPRC5D and CD3 according to any one of claims 1-12, wherein the second antigen-binding domain or the third antigen-binding domain comprises a Titin chain and an Obscurin chain capable of forming a dimer; Preferably, The titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 108 to SEQ ID NO:

126. The Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 127 to SEQ ID NO: 167; More preferably, The titin chain contains the amino acid sequence of SEQ ID NO: 126, and the obscurin chain contains the amino acid sequence of SEQ ID NO:

161.

14. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-13, wherein the antigen-binding molecule comprises a full-length antibody containing two heavy chains and two light chains, wherein, One heavy chain constant region CH1 of the full-length antibody is replaced by a Titin chain, and the light chain constant region CL that pairs with the heavy chain constant region CH1 is replaced by an Obscurin chain; or one heavy chain constant region CH1 of the full-length antibody is replaced by an Obscurin chain, and the light chain constant region CL that pairs with the heavy chain constant region CH1 is replaced by a Titin chain, wherein the Titin chain and the Obscurin chain pair with each other to form a dimer; Preferably, The full-length antibody contains a second antigen-binding domain that specifically binds to CD3 and a third antigen-binding domain that specifically binds to GPRC5D. More preferably, (i) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1 and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, the Obscurin chain and the Titin chain together form a replaced Fab that specifically binds to GPRC5D; (ii) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1 and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein VH, VL, obscurin chain and titin chain together form a replaced Fab that specifically binds to GPRC5D; (iii) One heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, the Obscurin chain, and the Titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab that specifically binds to GPRC5D; or (iv) One heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein the VH, VL, obscurin chain and titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains VL and a light chain constant region CL, wherein the VH, VL, CH1 and CL together form a Fab that specifically binds to GPRC5D; More preferably, One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1 and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, the Obscurin chain and the Titin chain together form a replaced Fab that specifically binds to GPRC5D.

15. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-14, wherein the antigen-binding molecule comprises a full-length antibody containing two heavy chains and two light chains, wherein, The N-terminus of the single variable domain of the immunoglobulin is operatively linked to the C-terminus of the light chain of the full-length antibody. Preferably, the first antigen-binding domain that specifically binds to BCMA comprises two immunoglobulin single variable domains, wherein the N-terminus of one immunoglobulin single variable domain is connected to the C-terminus of the constant region CL of the light chain of the full-length antibody, and the N-terminus of the other immunoglobulin single variable domain is connected to the C-terminus of the Obscurin chain or Titin chain on the light chain of the full-length antibody. More preferably, (i) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1 and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and an Obscurin chain, and the light chain bound to the heavy chain contains VL and a Titin chain, wherein VH, VL, Obscurin chain and Titin chain together form a replaced Fab that specifically binds to GPRC5D; the N-terminus of the two immunoglobulin single variable domains is respectively connected to the C-terminus of the light chain constant region CL and the C-terminus of the Titin chain on the light chain; (ii) One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein VH, VL, the obscurin chain, and the titin chain together form a replaced Fab that specifically binds to GPRC5D; the N-terminus of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the obscurin chain on the light chain, respectively; (iii) One heavy chain of the full-length antibody contains a VH and an Obscurin chain, and the light chain bound to the heavy chain contains a VL and a Titin chain, wherein the VH, VL, Obscurin chain, and Titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains a VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains a VL and a light chain constant region CL, wherein the VH, VL, CH1, and CL together form a Fab that specifically binds to GPRC5D; the N-terminus of each of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the Titin chain on the light chain, respectively; or (iv) One heavy chain of the full-length antibody contains VH and a titin chain, and the light chain bound to the heavy chain contains VL and an obscurin chain, wherein the VH, VL, obscurin chain, and titin chain together form a substituted Fab that specifically binds to CD3; the other heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and the light chain bound to the heavy chain contains VL and a light chain constant region CL, wherein the VH, VL, CH1, and CL together form a Fab that specifically binds to GPRC5D; the N-terminus of each of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the obscurin chain on the light chain, respectively; More preferably, The antigen-binding molecule comprises two immunoglobulin single variable domains. One heavy chain of the full-length antibody contains VH and a heavy chain constant region CH1, and one light chain contains VL and a light chain constant region CL, wherein VH, VL, CH1, and CL together form a Fab that specifically binds to CD3. The other heavy chain of the full-length antibody contains VH and an obscurin chain, and the light chain bound to the heavy chain contains VL and a titin chain, wherein VH, VL, the obscurin chain, and the titin chain together form a replaced Fab that specifically binds to GPRC5D. The N-terminus of the two immunoglobulin single variable domains is connected to the C-terminus of the light chain constant region CL and the C-terminus of the titin chain on the light chain, respectively.

16. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-15, comprising: A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d), wherein the structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end: (a) [GPRC5D-VL]-[Titin chain]-[connector 1]-[BCMA-VHH]; (b) [GPRC5D-VH]-[Obscurin chain]-[connector 2]-[Fc1]; (c)[CD3-VH]-[CH1]-[Fc2]; (d)[CD3-VL]-[CL]-[Connector 1]-[BCMA-VHH] The BCMA-VHH is a single variable domain of an immunoglobulin that specifically binds to BCMA; the CD3-VH and CD3-VL are VH and VL of the second antigen-binding domain that specifically binds to CD3; the GPRC5D-VH and GPRC5D-VL are VH and VL of the third antigen-binding domain that specifically binds to GPRC5D. The linker 1 and linker 2 may be the same or different peptide linkers, or linker 1 and linker 2 may not exist; The first and second chains combine with each other, the third and fourth chains combine with each other, and the second and third chains combine with each other to form antigen-binding molecules that specifically bind to BCMA, GPRC5D and CD3. Preferably, wherein, The BCMA-VHH contains the amino acid sequence of SEQ ID NO: 38; the GPRC5D-VH contains the amino acid sequence of SEQ ID NO: 79; the GPRC5D-VL contains the amino acid sequence of SEQ ID NO: 80; the CD3-VH contains the amino acid sequence of SEQ ID NO: 58; and the CD3-VL contains the amino acid sequence of SEQ ID NO: 59 or 60. More preferably, The BCMA-VHH contains the amino acid sequence of SEQ ID NO: 38; the GPRC5D-VH contains the amino acid sequence of SEQ ID NO: 79; the GPRC5D-VL contains the amino acid sequence of SEQ ID NO: 80; the CD3-VH contains the amino acid sequence of SEQ ID NO: 58; and the CD3-VL contains the amino acid sequence of SEQ ID NO:

59. Most preferably, The BCMA-VHH contains the amino acid sequence of SEQ ID NO: 38; the GPRC5D-VH contains the amino acid sequence of SEQ ID NO: 79; the GPRC5D-VL contains the amino acid sequence of SEQ ID NO: 80; the CD3-VH contains the amino acid sequence of SEQ ID NO: 58; and the CD3-VL contains the amino acid sequence of SEQ ID NO:

59. The titin chain contains the amino acid sequence of SEQ ID NO: 126; the obscurin chain contains the amino acid sequence of SEQ ID NO:

161.

17. The antigen-binding molecule that specifically binds BCMA, GPRC5D, and CD3 according to any one of claims 1-16, comprising a heavy chain constant region CH1 and a light chain constant region CL; wherein the heavy chain constant region CH1 is the heavy chain constant region CH1 of human IgG1, IgG2, IgG3, IgG4, or variants thereof, and the light chain constant region CL is the light chain constant region CL of human kappa or lambda. Preferably, the heavy chain constant region CH1 contains the amino acid sequence of SEQ ID NO: 89, and the light chain constant region CL contains the amino acid sequence of SEQ ID NO:

90.

18. The antigen-binding molecule that specifically binds BCMA, GPRC5D and CD3 according to any one of claims 1-17, comprising an Fc region, said Fc region being an IgG Fc region, said Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, said Fc1 and Fc2 each independently comprising one or more amino acid substitutions; Preferably, the Fc region is the IgG1 Fc region; Fc1 includes a protruding structure according to the pestle and mortar technique and Fc2 includes a hole structure according to the pestle and mortar technique; or Fc1 includes an F405L mutation and Fc2 includes a K409R mutation, numbered according to the EU index; More preferably, Fc1 contains the amino acid sequence of SEQ ID NO: 81, 82, 17 or 86; and Fc2 contains the amino acid sequence of SEQ ID NO: 84, 83, 88 or 87. More preferably, Fc1 and Fc2 are selected from the following group: Fc1 contains the amino acid sequence of SEQ ID NO: 81 and Fc2 contains the amino acid sequence of SEQ ID NO:

84. Fc1 contains the amino acid sequence of SEQ ID NO: 81 and Fc2 contains the amino acid sequence of SEQ ID NO:

83. Fc1 contains the amino acid sequence of SEQ ID NO: 82 and Fc2 contains the amino acid sequence of SEQ ID NO:

83. Fc1 contains the amino acid sequence of SEQ ID NO: 17 and Fc2 contains the amino acid sequence of SEQ ID NO:

88. Fc1 contains the amino acid sequence SEQ ID NO: 17 and Fc2 contains the amino acid sequence SEQ ID NO:

87. Fc1 contains the amino acid sequence of SEQ ID NO: 86 and Fc2 contains the amino acid sequence of SEQ ID NO: 87; Most preferably, Fc1 contains the amino acid sequence of SEQ ID NO: 81, and Fc2 contains the amino acid sequence of SEQ ID NO:

84.

19. The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 according to any one of claims 1-18, wherein the antigen-binding molecule specifically binds to BCMA, GPRC5D, and CD3 has: i) a first chain containing an amino acid sequence of SEQ ID NO: 92 or having at least 80% sequence identity with it, a second chain containing an amino acid sequence of SEQ ID NO: 93 or having at least 80% sequence identity with it, a third chain containing an amino acid sequence of SEQ ID NO: 94 or having at least 80% sequence identity with it, and a fourth chain containing an amino acid sequence of SEQ ID NO: 95 or having at least 80% sequence identity with it; or ii) A first chain containing an amino acid sequence of SEQ ID NO: 92 or having at least 80% sequence identity with it, a second chain containing an amino acid sequence of SEQ ID NO: 93 or having at least 80% sequence identity with it, a third chain containing an amino acid sequence of SEQ ID NO: 94 or having at least 80% sequence identity with it, and a fourth chain containing an amino acid sequence of SEQ ID NO: 96 or having at least 80% sequence identity with it; Preferably, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 has the following characteristics: i) A first chain containing the amino acid sequence of SEQ ID NO: 92, a second chain containing the amino acid sequence of SEQ ID NO: 93, a third chain containing the amino acid sequence of SEQ ID NO: 94, and a fourth chain containing the amino acid sequence of SEQ ID NO: 95; ii) A first chain containing the amino acid sequence of SEQ ID NO: 92, a second chain containing the amino acid sequence of SEQ ID NO: 93, a third chain containing the amino acid sequence of SEQ ID NO: 94, and a fourth chain containing the amino acid sequence of SEQ ID NO: 96; Preferably, the antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 has: a first chain containing the amino acid sequence of SEQ ID NO: 92, a second chain containing the amino acid sequence of SEQ ID NO: 93, a third chain containing the amino acid sequence of SEQ ID NO: 94, and a fourth chain containing the amino acid sequence of SEQ ID NO:

95.

20. An antigen-binding molecule that specifically binds to BCMA, comprising a single immunoglobulin variable domain, said single immunoglobulin variable domain comprising CDR1, CDR2, and CDR3 of the sequence SEQ ID NO: 25, 38, 22, 35, 23, 36, 24, 37, 26, 39, 27, or 40; or The immunoglobulin single variable domain includes CDR1, CDR2, and CDR3 in the sequence SEQ ID NO: 16 or 34.

21. An antigen-binding molecule that specifically binds to BCMA, comprising a single immunoglobulin variable domain comprising CDR1, CDR2, and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13, CDR2 comprises the amino acid sequence of SEQ ID NO: 168, and CDR3 comprises the amino acid sequence of SEQ ID NO:

15.

22. The antigen-binding molecule that specifically binds to BCMA according to claim 20 or 21, wherein the CDR1 of the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 13, the CDR2 comprises the amino acid sequence of SEQ ID NO: 14, 20, or 21, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 15; Preferably, The CDR1 of the immunoglobulin single variable domain contains the amino acid sequence of SEQ ID NO: 13, the CDR2 contains the amino acid sequence of SEQ ID NO: 14, and the CDR3 contains the amino acid sequence of SEQ ID NO:

15. The immunoglobulin's single variable domain CDR1 contains the amino acid sequence of SEQ ID NO: 13, CDR2 contains the amino acid sequence of SEQ ID NO: 20, and CDR3 contains the amino acid sequence of SEQ ID NO: 15; or The CDR1 of the immunoglobulin single variable domain contains the amino acid sequence of SEQ ID NO: 13, the CDR2 contains the amino acid sequence of SEQ ID NO: 21, and the CDR3 contains the amino acid sequence of SEQ ID NO:

15. More preferably, The immunoglobulin's single variable domain CDR1 contains the amino acid sequence of SEQ ID NO: 13, CDR2 contains the amino acid sequence of SEQ ID NO: 14, and CDR3 contains the amino acid sequence of SEQ ID NO:

15.

23. The antigen-binding molecule that specifically binds to BCMA according to any one of claims 20-22, wherein, The immunoglobulin's single variable domain is camel-derived, humanized, reverse-mutated, affinity-matured, T-cell epitope-removed, antibody deamidation reduced, and / or antibody isomerization reduced.

24. The antigen-binding molecule that specifically binds to BCMA according to any one of claims 20-23, wherein, The immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 169, 170, 171, 188, or 189.

25. The antigen-binding molecule that specifically binds to BCMA according to any one of claims 20-23, wherein the immunoglobulin single variable domain comprises SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27 or 40, or an amino acid sequence having at least 80% sequence identity with it; Preferably, The immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 25, 38, 16, 34, 22, 35, 23, 36, 24, 37, 26, 39, 27 or 40; More preferably, The immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 25, 38, 16, or 34; Most preferably, The immunoglobulin single variable domain contains the amino acid sequence of SEQ ID NO: 25 or 16.

26. The antigen-binding molecule that specifically binds to BCMA according to any one of claims 20 to 25, wherein the immunoglobulin single variable domain comprises an Fc region; Preferably, the Fc region is the Fc region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof; More preferably, the Fc region contains the amino acid sequence of SEQ ID NO: 85, 6 or 17.

27. The antigen-binding molecule that specifically binds to BCMA according to any one of claims 20 to 26, wherein the immunoglobulin single variable domain comprises SEQ ID NO: 31, 19, 28, 29, 30, 32 or 33, or an amino acid sequence having at least 80% sequence identity with it; Preferably, The immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 31, 19, 28, 29, 30, 32 or 33; More preferably, The immunoglobulin single variable domain contains the amino acid sequence of SEQ ID NO:

31.

28. The antigen-binding molecule that specifically binds to BCMA according to any one of claims 20 to 27, wherein the single variable domain of the immunoglobulin is an anti-BCMA nanobody or VHH.

29. The antigen-binding molecule that specifically binds to BCMA according to any one of claims 20 to 28, wherein, The antigen-binding molecule that specifically binds to BCMA is an anti-BCMA bispecific antibody. Preferably, The anti-BCMA bispecific antibody is a bispecific antibody that specifically binds to BCMA and CD3, comprising the immunoglobulin single variable domain and the antigen-binding domain that specifically binds to CD3 as described in any one of claims 22 to 28. More preferably, the antigen-binding domain that specifically binds to CD3 includes VH and VL, wherein VH includes HCDR1, HCDR2, and HCDR3, and VL includes LCDR1, LCDR2, and LCDR3, and the CDRs of the antigen-binding domain that specifically binds to CD3 are selected from the following group: The HCDR1 contains the amino acid sequence of SEQ ID NO: 41, the HCDR2 contains the amino acid sequence of SEQ ID NO: 42, the HCDR3 contains the amino acid sequence of SEQ ID NO: 43, the LCDR1 contains the amino acid sequence of SEQ ID NO: 44, the LCDR2 contains the amino acid sequence of SEQ ID NO: 45, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

46. The HCDR1 contains the amino acid sequence of SEQ ID NO: 47, the HCDR2 contains the amino acid sequence of SEQ ID NO: 48, the HCDR3 contains the amino acid sequence of SEQ ID NO: 49, and the LCDR1 contains the amino acid sequence of SEQ ID NO: 50, the LCDR2 contains the amino acid sequence of SEQ ID NO: 51, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 52; and The HCDR1 contains the amino acid sequence of SEQ ID NO: 47, the HCDR2 contains the amino acid sequence of SEQ ID NO: 48, the HCDR3 contains the amino acid sequence of SEQ ID NO: 49, the LCDR1 contains the amino acid sequence of SEQ ID NO: 53, the LCDR2 contains the amino acid sequence of SEQ ID NO: 54, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

55. More preferably, the variable region sequence of the antigen-binding domain that specifically binds to CD3 is selected from the following group: The VH contains the amino acid sequence of SEQ ID NO: 56 or an amino acid sequence having at least 80% sequence identity with it, and the VL contains the amino acid sequence of SEQ ID NO: 57 or an amino acid sequence having at least 80% sequence identity with it. The VH comprises the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence having at least 80% sequence identity with it, and the VL comprises the amino acid sequence of SEQ ID NO: 59 or an amino acid sequence having at least 80% sequence identity with it; and The VH contains the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence having at least 80% sequence identity with it, and the VL contains the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 80% sequence identity with it. Most preferably, the variable region sequence of the antigen-binding domain that specifically binds to CD3 is selected from the following group: The VH contains the amino acid sequence of SEQ ID NO: 56, and the VL contains the amino acid sequence of SEQ ID NO: 57; The VH contains the amino acid sequence of SEQ ID NO: 58, and the VL contains the amino acid sequence of SEQ ID NO: 59; and The VH contains the amino acid sequence of SEQ ID NO: 58, and the VL contains the amino acid sequence of SEQ ID NO:

60.

30. The antigen-binding molecule that specifically binds to BCMA according to claim 29, wherein the anti-BCMA bispecific antibody comprises a CD3-specific Fab domain, a BCMA-specific immunoglobulin single variable domain, and an Fc region, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, and the CD3-specific Fab domain and the BCMA-specific immunoglobulin single variable domain are operatively linked to the N-terminus of Fc1 and Fc2, respectively; Preferably, The anti-BCMA bispecific antibody comprises: A first chain with the structure shown in equation (e), a second chain with the structure shown in equation (f), and a third chain with the structure shown in equation (g), wherein the structures shown in equations (e), (f), and (g) are arranged from the N end to the C end: (e)[VHH]-[connector]-[Fc1]; (f)[VH]-[CH1]-[Fc2]; (g)[VL]-[CL]; The VHH is a single variable domain of an immunoglobulin that specifically binds to BCMA, and the VH and VL are VH and VL that specifically bind to the Fab domain of CD3. The linker is a peptide linker; the first chain and the second chain are linked together, and the third chain is linked together with the second chain to form a bispecific antibody that specifically binds to BCMA and CD3; More preferably, The heavy chain constant region CH1 is the heavy chain constant region CH1 of human IgG1 or its variants, the light chain constant region CL is the light chain constant region CL of human kappa or lambda, the Fc region is the IgG1 Fc region, and the Fc1 and Fc2 each independently contain one or more amino acid substitutions. More preferably, the bispecific antibody that specifically binds to BCMA and CD3 has the following characteristics: i) a first chain containing the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 80% sequence identity with it, a second chain containing the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 80% sequence identity with it, and a third chain containing the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 80% sequence identity with it. ii) A first chain containing the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 80% sequence identity with it, a second chain containing the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 80% sequence identity with it, and a third chain containing the amino acid sequence of SEQ ID NO: 67 or an amino acid sequence having at least 80% sequence identity with it. iii) A first chain containing the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 80% sequence identity with it, a second chain containing the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% sequence identity with it, and a third chain containing the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 80% sequence identity with it. iv) A first chain containing the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 80% sequence identity with it, a second chain containing the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% sequence identity with it, and a third chain containing the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 80% sequence identity with it. v) A first strand comprising the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 80% sequence identity with it, a second strand comprising the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% sequence identity with it, and a third strand comprising the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence having at least 80% sequence identity with it; or vi) A first chain containing the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence having at least 80% sequence identity with it, a second chain containing the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 80% sequence identity with it, and a third chain containing the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence having at least 80% sequence identity with it. The first chain and the second chain bind to each other, and the second chain and the third chain bind to each other to form a bispecific antibody that specifically binds to BCMA and CD3; Most preferably, The bispecific antibody that specifically binds to BCMA and CD3 has the following characteristics: i) A first strand containing the amino acid sequence of SEQ ID NO: 65, a second strand containing the amino acid sequence of SEQ ID NO: 66, and a third strand containing the amino acid sequence of SEQ ID NO: 67; ii) A first strand containing the amino acid sequence of SEQ ID NO: 68, a second strand containing the amino acid sequence of SEQ ID NO: 66, and a third strand containing the amino acid sequence of SEQ ID NO: 67; iii) A first strand containing the amino acid sequence of SEQ ID NO: 65, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 71; iv) A first strand containing the amino acid sequence of SEQ ID NO: 68, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 71; v) A first strand containing the amino acid sequence of SEQ ID NO: 65, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 72; or vi) A first strand containing the amino acid sequence of SEQ ID NO: 68, a second strand containing the amino acid sequence of SEQ ID NO: 70, and a third strand containing the amino acid sequence of SEQ ID NO: 72; The first chain and the second chain bind to each other, and the second chain and the third chain bind to each other to form a bispecific antibody that specifically binds to BCMA and CD3.

31. A pharmaceutical composition comprising: The antigen-binding molecule that specifically binds to BCMA, GPRC5D, and CD3 as described in any one of claims 1 to 19, or the antigen-binding molecule that specifically binds to BCMA as described in any one of claims 20 to 30, and One or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

32. Nucleic acid, its encoding: The antigen-binding molecule that specifically binds to BCMA, GPRC5D and CD3 as described in any one of claims 1 to 19, or the antigen-binding molecule that specifically binds to BCMA as described in any one of claims 20 to 30.

33. A host cell comprising the nucleic acid of claim 32.

34. A method for treating a disease or symptom, the method comprising: The subject is given a therapeutically effective amount of the antigen-binding molecule that specifically binds to BCMA, GPRC5D and CD3 as described in any one of claims 1 to 19, or the antigen-binding molecule that specifically binds to BCMA as described in any one of claims 20 to 30, or the pharmaceutical composition as described in claim 31. Preferably, the disease or symptom is cancer; More preferably, the disease or symptom is a hematoma; More preferably, the disease or condition is B-cell carcinoma expressing BCMA and / or GPRC5D; More preferably, the disease or symptom is multiple myeloma; Most preferably, the disease or condition is relapsed or refractory multiple myeloma.

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