Multispecific antigen-binding protein and use thereof

Abstract

Provided are a multispecific antigen-binding protein and a use thereof. Specifically provided is a bispecific or trispecific antigen-binding protein formed by linking scFv or Fab fragments derived from an anti-GPRC5D antibody, an anti-BCMA antibody, and an anti-CD3 antibody. The multispecific antigen-binding protein simultaneously targets two or three targets among GPRC5D, BCMA, and CD3, thereby improving the therapeutic effect against tumors; the present invention has application prospects in the anti-tumor field.

Description

A multispecific antigen-binding protein and its applications Technical Field

[0001] This invention relates to the field of antibody drugs, and more specifically, to a multispecific antigen-binding protein and its applications. Background Technology

[0002] Currently, the main treatment options for cancer patients are still surgery, traditional chemotherapy, radiotherapy, or small molecule targeted drugs. However, with the gradual application of immunotherapy, especially the publication of groundbreaking results from various clinical trials, cancer immunotherapy has become a new treatment method, bringing hope to cancer patients.

[0003] Current cancer immunotherapy strategies mainly focus on enhancing the activation (exogenous or endogenous) of effector T cells in cancer patients, releasing suppressed anti-cancer T cell responses, or breaking the tolerance of T cells to tumor cells, thereby killing and eliminating tumor cells to achieve the goal of treating patients.

[0004] Currently, immunotherapy for cancer patients mainly includes: 1) checkpoint inhibitors (CPI), 2) chimeric antigen receptor T cells (CAR-T), 3) antibody-drug conjugates (ADC), and 4) T cell engagers (TCE), etc.

[0005] T-cell engagers are artificially designed molecules that specifically bind to two or more different antigens, with one end binding to a CD3 antigen and the other end binding to one or more tumor-associated antigens (TAAs) and / or tumor-specific antigens (TSAs). These T-cell engagers cross-link tumor cells and T cells, forming a T-cell engager that leads to synapse formation at the contact site, activating cytotoxic signals and causing tumor cell lysis. When T cells are activated, the cell surface markers CD25 and CD69 are upregulated, and cytokines are released. These secreted cytokines, such as interleukin-2 (IL-2), further activate T cells and promote their proliferation, enhancing their ability to kill tumor cells.

[0006] GPRC5D is a G protein-coupled receptor (GPCR), short for G Protein-Coupled Receptor, Class C Group 5 Member D. It is a transmembrane protein and belongs to Group C of the G protein-coupled receptor family. GPRC5D is expressed at low levels in normal tissues, but its expression is elevated in certain cancers such as multiple myeloma (MM).

[0007] The detailed physiological functions of GPRC5D are not yet fully understood. As a G protein-coupled receptor, it may participate in intracellular signal transduction and regulate a variety of biological processes by binding to ligands and activating downstream G protein signaling pathways. It may be involved in cell growth, differentiation, and other key physiological and pathological processes.

[0008] Aberrant expression of GPRC5D is associated with the development and progression of diseases such as multiple myeloma. In MM, overexpression of GPRC5D may be related to tumor cell proliferation, survival, and drug resistance.

[0009] B cell maturation antigen (BCMA) is a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily (TNFRSF). BCMA is primarily expressed on mature B cells and plays a crucial role in the B cell life cycle. It regulates B cell survival, proliferation, and antibody secretion by binding to its two main ligands, APRIL (proliferation-inducing ligand) and BAFF (B cell activating factor).

[0010] In B-cell malignancies such as multiple myeloma, BCMA expression is significantly increased, which is closely related to tumor cell proliferation and survival. Tumor cells can activate BCMA through autocrine secretion of APRIL and BAFF, thereby promoting self-proliferation and evading immune system surveillance. High BCMA expression is also associated with disease progression and poor prognosis, thus becoming a clinically significant biomarker.

[0011] BCMA's unique expression pattern and crucial role in the progression of B-cell malignancies make it an ideal therapeutic target. BCMA-targeted therapies can achieve specific attacks on diseased B cells with relatively minimal impact on normal tissues. Furthermore, BCMA-targeted therapies can exert their effects through multiple mechanisms, including directly inducing tumor cell apoptosis, blocking tumor growth signaling, and activating the immune system's attack on tumor cells.

[0012] The independent expression of GPRC5D and BCMA opens up possibilities for the development of single- or dual-targeted therapies. Targeted therapy with GPRC5D aims to address the relapse of multiple myeloma (MM) caused by BCMA escape and holds promise for improving the efficacy of current antibody or T-cell therapies that target BCMA only.

[0013] Despite the significant clinical success of GPRC5D or BCMA-targeted therapies, such as CAR-T therapy, their complex manufacturing processes and high costs limit their widespread application. Several unresolved issues remain in clinical practice, including resistance to GPRC5D or BCMA-targeted therapies in some patients, improving long-term survival rates, and optimizing efficacy in children and the elderly.

[0014] With a deeper understanding of the tumor microenvironment and tumor immunology, future treatments may include combinations of targeted therapies with other treatment modalities, such as radiotherapy, chemotherapy, immunomodulators, or other emerging targeted therapies. For example, simultaneously targeting GPRRC5D and BCMA can avoid antigen escape from a single target; this multimodal treatment strategy may improve treatment outcomes and provide patients with more comprehensive treatment options. Summary of the Invention

[0015] The purpose of this invention is to provide a multispecific antigen-binding protein capable of binding GPRC5D, BCMA and CD3 and its applications.

[0016] In a first aspect of the invention, a multispecific antigen-binding protein is provided, the antigen-binding protein comprising at least two antigen-binding domains selected from the group consisting of: (Z1) one or more CD3-binding domains, (Z2) one or more GPRC5D-binding domains, and (Z3) one or more BCMA-binding domains.

[0017] In another preferred embodiment, the antigen-binding domain is in the form of a Fab fragment, a single-chain Fv (scFv), an F(ab')2 fragment, a single-domain antibody (sdAb), or a combination thereof.

[0018] In another preferred embodiment, the antigen-binding domain binding GPRC5D is a Fab fragment, the antigen-binding domain binding CD3 is an scFv fragment, and / or the antigen-binding domain binding BCMA is an scFv fragment.

[0019] In another preferred embodiment, the antigen-binding domain binding CD3 is a Fab fragment, the antigen-binding domain binding GPRC5D is an scFv fragment, and / or the antigen-binding domain binding BCMA is an scFv fragment.

[0020] In another preferred embodiment, the antigen-binding domain binding BCMA is a Fab fragment, the antigen-binding domain binding CD3 is an scFv fragment, and / or the antigen-binding domain binding GPRC5D is an scFv fragment.

[0021] In another preferred embodiment, the antigen-binding domain that binds to GPRC5D comprises the following three heavy chain variable regions (CDRs):

[0022] HCDR1 has an amino acid sequence as shown in SEQ ID NO:22 or 25;

[0023] HCDR2, which has the amino acid sequence shown in SEQ ID NO:23; and

[0024] HCDR3 has an amino acid sequence as shown in SEQ ID NO:24 or 26;

[0025] And the following three light chain variable regions (CDRs):

[0026] LCDR1 has the amino acid sequence shown in SEQ ID NO:27;

[0027] LCDR2, which has the amino acid sequence shown in SEQ ID NO:28; and

[0028] LCDR3 has the amino acid sequence shown in SEQ ID NO:29.

[0029] In another preferred embodiment, the antigen-binding domain that binds GPRC5D includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1, 2, or 3.

[0030] In another preferred embodiment, the antigen-binding domain that binds GPRC5D includes a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0031] In another preferred embodiment, the antigen-binding domain that binds to GPRC5D includes a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:4.

[0032] In another preferred embodiment, the antigen-binding domain that binds GPRC5D includes a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:4.

[0033] In another preferred embodiment, the antigen-binding domain that binds to GPRC5D includes a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:4.

[0034] In another preferred embodiment, the antigen-binding domain that binds to GPRC5D is selected from the group consisting of scFv, Fab, or combinations thereof.

[0035] In another preferred embodiment, the antigen-binding domain that binds to GPRC5D is Fab, which includes the heavy chain variable region and the light chain variable region as described above, as well as the heavy chain constant region CH1 and the light chain constant region CL.

[0036] In another preferred embodiment, the antigen-binding domain that binds to GPRC5D is Fab, which includes a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:1, 2, or 3, and a light chain variable region of an amino acid sequence as shown in SEQ ID NO:4.

[0037] And the heavy chain constant region CH1 of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region CL of the amino acid sequence shown in SEQ ID NO:10.

[0038] In another preferred embodiment, the antigen-binding domain that binds to BCMA comprises the following three heavy chain variable regions (CDRs):

[0039] HCDR1 has the amino acid sequence shown in SEQ ID NO:30;

[0040] HCDR2, which has the amino acid sequence shown in SEQ ID NO:31; and

[0041] HCDR3 has the amino acid sequence shown in SEQ ID NO:32;

[0042] And the following three light chain variable regions (CDRs):

[0043] LCDR1 has the amino acid sequence shown in SEQ ID NO:33;

[0044] LCDR2, which has the amino acid sequence shown in SEQ ID NO:34; and

[0045] LCDR3 has the amino acid sequence shown in SEQ ID NO:35.

[0046] In another preferred embodiment, the antigen-binding domain that binds BCMA includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7.

[0047] In another preferred embodiment, the antigen-binding domain that binds BCMA includes a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0048] In another preferred embodiment, the antigen-binding domain that binds to BCMA includes a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0049] In another preferred embodiment, the antigen-binding domain that binds to BCMA is selected from the group consisting of scFv, Fab, or combinations thereof.

[0050] In another preferred embodiment, the antigen-binding domain that binds to BCMA is scFv.

[0051] In another preferred embodiment, the CD3-binding antigen-binding domain comprises the following three heavy chain variable regions (CDRs):

[0052] HCDR1 has the amino acid sequence shown in SEQ ID NO:44;

[0053] HCDR2, which has the amino acid sequence shown in SEQ ID NO:45; and

[0054] HCDR3 has the amino acid sequence shown in SEQ ID NO:46;

[0055] And the following three light chain variable regions (CDRs):

[0056] LCDR1 has the amino acid sequence shown in SEQ ID NO:47;

[0057] LCDR2, which has the amino acid sequence shown in SEQ ID NO:48; and

[0058] LCDR3 has the amino acid sequence shown in SEQ ID NO:49.

[0059] In another preferred embodiment, the antigen-binding domain that binds CD3 includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5.

[0060] In another preferred embodiment, the antigen-binding domain that binds CD3 includes a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6.

[0061] In another preferred embodiment, the antigen-binding domain that binds to CD3 includes a heavy chain variable region as shown in SEQ ID NO:5 and a light chain variable region as shown in SEQ ID NO:6.

[0062] In another preferred embodiment, the antigen-binding domain that binds to CD3 is selected from the group consisting of scFv, Fab, or combinations thereof.

[0063] In another preferred embodiment, the antigen-binding domain that binds to CD3 is scFv.

[0064] In another preferred embodiment, the antigen-binding domain that binds to CD3 is Fab.

[0065] In another preferred embodiment, the antigen-binding domain that binds to CD3 is Fab, which includes the heavy chain variable region and the light chain variable region as described above, as well as the heavy chain constant region CH1 of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region CL of the amino acid sequence shown in SEQ ID NO:36.

[0066] In another preferred embodiment, the multispecific antigen-binding protein is a bispecific antigen-binding protein (or a bispecific antibody or a bispecific T-cell connector).

[0067] In another preferred embodiment, the bispecific antigen-binding protein comprises one or more (e.g., 2, 3, or 4) antigen-binding domains that bind GPRC5D and one or more (e.g., 2, 3, or 4) antigen-binding domains that bind CD3.

[0068] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds GPRC5D and an antigen-binding domain that binds CD3.

[0069] In another preferred embodiment, the antigen-binding domain that binds GPRC5D is Fab, and the antigen-binding domain that binds CD3 is scFv.

[0070] In another preferred embodiment, the antigen-binding domain that binds GPRC5D is scFv, and the antigen-binding domain that binds CD3 is Fab.

[0071] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds GPRC5D and an antigen-binding domain that binds CD3; wherein the antigen-binding domain that binds GPRC5D is Fab and the antigen-binding domain that binds CD3 is scFv.

[0072] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds GPRC5D and an antigen-binding domain that binds CD3; wherein the antigen-binding domain that binds GPRC5D is scFv and the antigen-binding domain that binds CD3 is Fab.

[0073] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds GPRC5D and two antigen-binding domains that bind CD3; wherein the antigen-binding domain that binds GPRC5D is Fab and the antigen-binding domain that binds CD3 is scFv.

[0074] In another preferred embodiment, the bispecific antigen-binding protein comprises two antigen-binding domains that bind GPRC5D and one antigen-binding domain that binds CD3; wherein the antigen-binding domain that binds GPRC5D is scFv and the antigen-binding domain that binds CD3 is Fab.

[0075] In another preferred embodiment, in the bispecific antigen-binding protein, the CD3-binding antigen-binding domain is linked to the C-terminus or N-terminus of the GPRC5D-binding antigen-binding domain; preferably, the linkage is made by a flexible linker peptide.

[0076] In another preferred embodiment, the linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer from 1 to 6, preferably 1, 2, or 3.

[0077] In another preferred embodiment, the bispecific antigen-binding protein has the structure shown in Formula I:

[0078] In the formula, each "-" represents a bond or linking peptide independently;

[0079] VH GPRC5D For binding the antigen-binding domain Fab of GPRC5D;

[0080] VL GPRC5D For binding the antigen-binding domain Fab of GPRC5D to the light chain variable region;

[0081] CH1 is the heavy chain constant region 1;

[0082] CL represents the constant region of the light chain;

[0083] scFv CD3 It is the antigen-binding domain that binds to CD3;

[0084] It is a disulfide bond or a covalent bond.

[0085] In another preferred embodiment, the VH GPRC5D Having an amino acid sequence as shown in SEQ ID NO:1, 2 or 3, and VL GPRC5D It has the amino acid sequence shown in SEQ ID NO:4.

[0086] In another preferred embodiment, the scFv CD3 It has the following structure:

[0087] VH CD3 -VL CD3 (A); or

[0088] VL CD3 -VH CD3 (B);

[0089] Among them, VH CD3 The heavy chain variable region that binds to the antigen-binding domain of CD3; VL CD3 The light chain variable region that binds to the antigen-binding domain of CD3

[0090] Preferably, it has the structure shown in formula (A).

[0091] In another preferred embodiment, the scFv CD3 The amino acid sequence has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:11.

[0092] In another preferred embodiment, the scFv CD3 The amino acid sequence is shown in SEQ ID NO:11.

[0093] In another preferred embodiment, the "VH" GPRC5D -CH1-scFv CD3 The amino acid sequence of the "VL" (i.e., heavy chain) is shown in SEQ ID NO:13, 15 or 16. GPRC5D The amino acid sequence of the "-CL" (i.e., light chain) is shown in SEQ ID NO:14.

[0094] In another preferred embodiment, the bispecific antigen-binding protein comprises one or more (e.g., 2, 3, or 4) antigen-binding domains that bind BCMA and one or more (e.g., 2, 3, or 4) antigen-binding domains that bind CD3.

[0095] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds BCMA and an antigen-binding domain that binds CD3.

[0096] In another preferred embodiment, the antigen-binding domain that binds BCMA is scFv, and the antigen-binding domain that binds CD3 is Fab.

[0097] In another preferred embodiment, the antigen-binding domain that binds BCMA is Fab, and the antigen-binding domain that binds CD3 is scFv.

[0098] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds BCMA and an antigen-binding domain that binds CD3; wherein the antigen-binding domain that binds BCMA is scFv and the antigen-binding domain that binds CD3 is Fab.

[0099] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds BCMA and an antigen-binding domain that binds CD3; wherein the antigen-binding domain that binds BCMA is Fab and the antigen-binding domain that binds CD3 is scFv.

[0100] In another preferred embodiment, the bispecific antigen-binding protein comprises two antigen-binding domains that bind BCMA and one antigen-binding domain that binds CD3; wherein the antigen-binding domain that binds BCMA is scFv and the antigen-binding domain that binds CD3 is Fab.

[0101] In another preferred embodiment, the bispecific antigen-binding protein comprises an antigen-binding domain that binds BCMA and two antigen-binding domains that bind CD3; wherein the antigen-binding domain that binds BCMA is Fab and the antigen-binding domain that binds CD3 is scFv.

[0102] In another preferred embodiment, in the bispecific antigen-binding protein, the CD3-binding antigen-binding domain is linked to the C-terminus or N-terminus of the BCMA-binding antigen-binding domain; preferably, the linkage is made by a flexible linker peptide.

[0103] In another preferred embodiment, the linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer from 1 to 6, preferably 1, 2, or 3.

[0104] In another preferred embodiment, the bispecific antigen-binding protein has the structure shown in Formula II:

[0105] In the formula, each "-" represents a bond or linking peptide independently;

[0106] VH CD3 For the heavy chain variable region of Fab, which binds to the antigen-binding domain of CD3;

[0107] VL CD3 For the light chain variable region of Fab, which binds to the antigen-binding domain of CD3;

[0108] CH1 is the heavy chain constant region 1;

[0109] CL represents the constant region of the light chain;

[0110] scFv BCMA To bind the antigen-binding domain of BCMA;

[0111] It is a disulfide bond or a covalent bond.

[0112] In another preferred embodiment, the VH CD3 It has the amino acid sequence shown in SEQ ID NO:5, and VL CD3 It has the amino acid sequence shown in SEQ ID NO:6.

[0113] In another preferred embodiment, the scFv BCMA It has the following structure:

[0114] VH BCMA -VL BCMA (C); or

[0115] VL BCMA -VH BCMA (D);

[0116] Among them, VH BCMA The heavy chain variable region, which binds to the antigen-binding domain of BCMA; VL BCMA The light chain variable region that binds to the antigen-binding domain of BCMA

[0117] Preferably, it has the structure shown in formula (D).

[0118] In another preferred embodiment, the scFv BCMA The amino acid sequence has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0119] In another preferred embodiment, the scFv BCMA The amino acid sequence is shown in SEQ ID NO:12.

[0120] In another preferred embodiment, the "VH" CD3 The amino acid sequence of "-CH1" (i.e., the heavy chain) is shown in SEQ ID NO:18, and the "scFv" BCMA -VL CD3 The amino acid sequence of the "-CL" (i.e., light chain) is shown in SEQ ID NO:19.

[0121] In another preferred embodiment, the multispecific antigen-binding protein is a trispecific antigen-binding protein (or a trispecific antibody or a trispecific T-cell connector).

[0122] In another preferred embodiment, the trispecific antigen-binding protein comprises one or more (e.g., 2, 3, or 4) antigen-binding domains that bind GPRC5D, one or more (e.g., 2, 3, or 4) antigen-binding domains that bind CD3, and one or more (e.g., 2, 3, or 4) antigen-binding domains that bind BCMA.

[0123] In another preferred embodiment, the antigen-binding domain binding GPRC5D is Fab, the antigen-binding domain binding CD3 is scFv, and the antigen-binding domain binding BCMA is scFv.

[0124] In another preferred embodiment, the antigen-binding domain binding GPRC5D is scFv, the antigen-binding domain binding CD3 is Fab, and the antigen-binding domain binding BCMA is scFv.

[0125] In another preferred embodiment, the antigen-binding domain that binds GPRC5D is scFv, the antigen-binding domain that binds CD3 is scFv, and the antigen-binding domain that binds BCMA is Fab.

[0126] In another preferred embodiment, the trispecific antigen-binding protein comprises an antigen-binding domain that binds GPRC5D, an antigen-binding domain that binds CD3, and an antigen-binding domain that binds BCMA; wherein the antigen-binding domain that binds GPRC5D is Fab, the antigen-binding domain that binds CD3 is scFv, and the antigen-binding domain that binds BCMA is scFv.

[0127] In another preferred embodiment, the trispecific antigen-binding protein comprises an antigen-binding domain that binds GPRC5D, an antigen-binding domain that binds CD3, and an antigen-binding domain that binds BCMA; wherein the antigen-binding domain that binds GPRC5D is scFv, the antigen-binding domain that binds CD3 is Fab, and the antigen-binding domain that binds BCMA is scFv.

[0128] In another preferred embodiment, the trispecific antigen-binding protein comprises an antigen-binding domain that binds GPRC5D, an antigen-binding domain that binds CD3, and an antigen-binding domain that binds BCMA; wherein the antigen-binding domain that binds GPRC5D is scFv, the antigen-binding domain that binds CD3 is scFv, and the antigen-binding domain that binds BCMA is Fab.

[0129] In another preferred embodiment, in the trispecific antigen-binding protein, the CD3-binding antigen-binding domain is attached to the C-terminus of the GPRC5D-binding antigen-binding domain, and the BCMA-binding antigen-binding domain is attached to the N-terminus of the GPRC5D-binding antigen-binding domain; or, the CD3-binding antigen-binding domain is attached to the N-terminus of the GPRC5D-binding antigen-binding domain, and the BCMA-binding antigen-binding domain is attached to the C-terminus of the GPRC5D-binding antigen-binding domain.

[0130] Alternatively, the antigen-binding domain that binds GPRC5D is attached to the C-terminus of the antigen-binding domain that binds CD3, and the antigen-binding domain that binds BCMA is attached to the N-terminus of the antigen-binding domain that binds CD3; or, the antigen-binding domain that binds BCMA is attached to the C-terminus of the antigen-binding domain that binds CD3, and the antigen-binding domain that binds GPRC5D is attached to the N-terminus of the antigen-binding domain that binds CD3.

[0131] Alternatively, the CD3-binding antigen-binding domain is attached to the C-terminus of the BCMA-binding antigen-binding domain, and the GPRC5D-binding antigen-binding domain is attached to the N-terminus of the BCMA-binding antigen-binding domain; or, the GPRC5D-binding antigen-binding domain is attached to the C-terminus of the BCMA-binding antigen-binding domain, and the CD3-binding antigen-binding domain is attached to the N-terminus of the BCMA-binding antigen-binding domain.

[0132] Preferably, the linkage is achieved via a flexible linker peptide.

[0133] In another preferred embodiment, the linker peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer from 1 to 6, preferably 1, 2, or 3.

[0134] In another preferred embodiment, the trispecific antigen-binding protein has the structure shown in Formula III:

[0135] In the formula, each "-" represents a bond or linking peptide independently;

[0136] VH GPRC5D For binding the antigen-binding domain Fab of GPRC5D;

[0137] VL GPRC5D For binding the antigen-binding domain Fab of GPRC5D to the light chain variable region;

[0138] CH1 is the heavy chain constant region 1;

[0139] CL represents the constant region of the light chain;

[0140] scFv CD3 It is the antigen-binding domain that binds to CD3;

[0141] scFv BCMA To bind the antigen-binding domain of BCMA;

[0142] It is a disulfide bond or a covalent bond.

[0143] In another preferred embodiment, the VH GPRC5D Having an amino acid sequence as shown in SEQ ID NO:1, 2 or 3, and VL GPRC5D It has the amino acid sequence shown in SEQ ID NO:4.

[0144] In another preferred embodiment, the scFv CD3 It has the following structure:

[0145] VH CD3 -VL CD3 (A); or

[0146] VL CD3 -VH CD3 (B);

[0147] Among them, VH CD3 The heavy chain variable region that binds to the antigen-binding domain of CD3; VL CD3 The light chain variable region that binds to the antigen-binding domain of CD3

[0148] Preferably, it has the structure shown in formula (A).

[0149] In another preferred embodiment, the scFv CD3 The amino acid sequence has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:11.

[0150] In another preferred embodiment, the scFv CD3 The amino acid sequence is shown in SEQ ID NO:11.

[0151] In another preferred embodiment, the scFv BCMA It has the following structure:

[0152] VH BCMA -VL BCMA (C); or

[0153] VL BCMA -VH BCMA (D);

[0154] Among them, VH BCMA The heavy chain variable region, which binds to the antigen-binding domain of BCMA; VL BCMA The light chain variable region that binds to the antigen-binding domain of BCMA

[0155] Preferably, it has the structure shown in formula (D).

[0156] In another preferred embodiment, the scFv BCMA The amino acid sequence has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0157] In another preferred embodiment, the scFv BCMA The amino acid sequence is shown in SEQ ID NO:12.

[0158] In another preferred embodiment, the "VH" GPRC5D -CH1-scFv CD3 The amino acid sequence of the "scFv" (i.e., the heavy chain) is shown in SEQ ID NO:13, 15 or 16. BCMA -VL GPRC5D The amino acid sequence of the "-CL" (i.e., light chain) is shown in SEQ ID NO:17.

[0159] A second aspect of the present invention provides an anti-GPRC5D antibody or an antigen-binding fragment thereof, said antibody comprising a heavy chain variable region and a light chain variable region.

[0160] The heavy chain variable region has the following complementary determining regions (CDRs):

[0161] HCDR1 as shown in SEQ ID NO:22 or 25

[0162] HCDR2 shown in SEQ ID NO:23, and

[0163] HCDR3 as shown in SEQ ID NO:24 or 26;

[0164] Furthermore, the light chain variable region has the following complementary determinant region (CDR):

[0165] LCDR1 shown in SEQ ID NO:27

[0166] LCDR2 shown in SEQ ID NO:28, and

[0167] LCDR3 as shown in SEQ ID NO:29.

[0168] In another preferred embodiment, the heavy chain variable region of the antibody has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1, 2, or 3.

[0169] In another preferred embodiment, the light chain variable region of the antibody has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0170] In another preferred embodiment, the antibody comprises a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:4.

[0171] In another preferred embodiment, the antibody comprises a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:4.

[0172] In another preferred embodiment, the antibody comprises a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:4.

[0173] In another preferred embodiment, the antigen-binding fragment is selected from the group consisting of Fab, (Fab')2, scFv, or combinations thereof.

[0174] A third aspect of the present invention provides an anti-BCMA antibody or an antigen-binding fragment thereof, said antibody comprising a heavy chain variable region and a light chain variable region.

[0175] The heavy chain variable region has the following complementary determining regions (CDRs):

[0176] HCDR1, as shown in SEQ ID NO:30,

[0177] HCDR2 shown in SEQ ID NO:31, and

[0178] HCDR3 as shown in SEQ ID NO:32;

[0179] Furthermore, the light chain variable region has the following complementary determinant region (CDR):

[0180] LCDR1 shown in SEQ ID NO:33

[0181] LCDR2 shown in SEQ ID NO:34, and

[0182] LCDR3 as shown in SEQ ID NO:35.

[0183] In another preferred embodiment, the heavy chain variable region of the antibody has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7.

[0184] In another preferred embodiment, the light chain variable region of the antibody comprises a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0185] In another preferred embodiment, the antibody comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:8.

[0186] In another preferred embodiment, the antigen-binding fragment is selected from the group consisting of Fab, (Fab')2, scFv, or combinations thereof.

[0187] A fourth aspect of the present invention provides a recombinant protein comprising:

[0188] (i) a multispecific antigen-binding protein as described in the first aspect of the invention, an anti-GPRC5D antibody or its antigen-binding fragment as described in the second aspect of the invention, or an anti-BCMA antibody or its antigen-binding fragment as described in the third aspect of the invention; and

[0189] (ii) Tag sequences that assist in expression and / or purification.

[0190] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, a FLAG tag, and / or a CH1 tag.

[0191] In another preferred embodiment, the recombinant protein is a fusion protein.

[0192] In another preferred embodiment, the recombinant protein is a monomer.

[0193] In a fifth aspect, the present invention provides a polynucleotide encoding a multispecific antigen-binding protein as described in the first aspect of the present invention, an anti-GPRC5D antibody or an antigen-binding fragment thereof as described in the second aspect of the present invention, an anti-BCMA antibody or an antigen-binding fragment thereof as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention.

[0194] In another preferred embodiment, the polynucleotide includes DNA and RNA.

[0195] In a sixth aspect, the present invention provides a carrier containing a polynucleotide as described in the fifth aspect of the present invention.

[0196] In another preferred embodiment, the carrier is an expression carrier.

[0197] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0198] In another preferred embodiment, the expression vector is a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0199] In a seventh aspect, the present invention provides a genetically engineered host cell containing a vector as described in the sixth aspect of the present invention, or having an exogenous polynucleotide as described in the fifth aspect of the present invention integrated into its genome.

[0200] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0201] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0202] A seventh aspect of the present invention provides an antibody conjugate comprising:

[0203] (a) An antibody portion, said antibody portion being selected from the group consisting of: multispecific antigen-binding proteins as described in the first aspect of the present invention, anti-GPRC5D antibodies or antigen-binding fragments thereof as described in the second aspect of the present invention, anti-BCMA antibodies or antigen-binding fragments thereof as described in the third aspect of the present invention, or recombinant proteins as described in the fourth aspect of the present invention; and

[0204] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of detectable markers, drugs, or combinations thereof.

[0205] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0206] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0207] A ninth aspect of the present invention provides the use of an active ingredient in the preparation of a medicament for the prevention and / or treatment of diseases associated with GPRC5D and / or BCMA expression, said active ingredient being selected from the group consisting of: multispecific antigen-binding proteins as described in the first aspect of the present invention, anti-GPRC5D antibodies or antigen-binding fragments thereof as described in the second aspect of the present invention, anti-BCMA antibodies or antigen-binding fragments thereof as described in the third aspect of the present invention, recombinant proteins as described in the fourth aspect of the present invention, or antibody conjugates as described in the eighth aspect of the present invention, or combinations thereof.

[0208] In another preferred embodiment, the disease is cancer or tumor.

[0209] In another preferred embodiment, the cancer or tumor is a solid tumor or a hematoma.

[0210] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0211] A tenth aspect of the present invention provides a pharmaceutical composition comprising...

[0212] (i) An active ingredient selected from the group consisting of: a multispecific antigen-binding protein as described in the first aspect of the present invention, an anti-GPRC5D antibody or an antigen-binding fragment thereof as described in the second aspect of the present invention, an anti-BCMA antibody or an antigen-binding fragment thereof as described in the third aspect of the present invention, a recombinant protein as described in the fourth aspect of the present invention, or an antibody conjugate as described in the eighth aspect of the present invention, or a combination thereof.

[0213] (ii) Pharmaceutically acceptable carriers.

[0214] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0215] In another preferred embodiment, the pharmaceutical composition is an injection.

[0216] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the active ingredient and 0.01 to 99.99% of the pharmaceutical carrier, wherein the percentages are percentages by mass of the pharmaceutical composition.

[0217] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of diseases associated with GPRC5D and / or BCMA expression.

[0218] In another preferred embodiment, the disease associated with GPRC5D and / or BCMA expression is cancer or tumor.

[0219] In another preferred embodiment, the cancer or tumor is a solid tumor or a hematoma.

[0220] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0221] The eleventh aspect of the present invention provides the use of a multispecific antigen-binding protein as described in the first aspect of the present invention, an anti-GPRC5D antibody or its antigen-binding fragment as described in the second aspect of the present invention, an anti-BCMA antibody or its antigen-binding fragment as described in the third aspect of the present invention, a recombinant protein as described in the fourth aspect of the present invention, and / or an antibody-drug conjugate as described in the eighth aspect of the present invention in the preparation of a reagent for detecting diseases associated with GPRC5D and / or BCMA expression.

[0222] In another preferred embodiment, the disease associated with GPRC5D and / or BCMA expression is cancer or tumor.

[0223] In another preferred embodiment, the reagent is used as an in vitro detection reagent or for in vivo detection.

[0224] A twelfth aspect of the present invention provides a method for treating a disease associated with GPRC5D and / or BCMA expression, comprising administering to a subject in need an effective amount of a multispecific antigen-binding protein as described in the first aspect of the present invention, an anti-GPRC5D antibody or an antigen-binding fragment thereof as described in the second aspect of the present invention, an anti-BCMA antibody or an antigen-binding fragment thereof as described in the third aspect of the present invention, a recombinant protein as described in the fourth aspect of the present invention, an antibody-drug conjugate as described in the eighth aspect of the present invention, or a pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof.

[0225] In another preferred embodiment, the disease is cancer or tumor.

[0226] In another preferred embodiment, the cancer or tumor is a solid tumor or a hematoma.

[0227] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0228] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0229] Figure 1 shows a schematic diagram of the structure of the heavy chain transient expression vector.

[0230] Figure 2 shows a schematic diagram of the structure of the light chain transient expression vector.

[0231] Figure 3 shows the cell binding curves of the constructed bispecific antibody molecules IMP-2023-041 and IMP-2023-042.

[0232] Figure 4 shows the cell binding curves of the trispecific antibody molecule IMP-2023-045. A: T cell binding curve; BD: 293F-GPRC5D, K562-BCMA, and NCI-H929 cell binding curves, respectively.

[0233] Figure 5 shows the cell killing curves of the CD3 x GPRC5D series of molecules; where the effector cells of A and B are Pri-T cells, the target cells are NCI-H929 and 293F-GPRC5D, respectively, the E:T ratio is 4:1, and the incubation time is 24 hours.

[0234] Figure 6 shows the molecular cell killing curves of the IMP-2023 series; where the effector cells of A and B are hPBMCs, the target cells are NCI-H929 and 293F-GPRC5D, respectively, the E:T ratio is 10:1, and the incubation time is 24 hours.

[0235] Figure 7 shows the target cell killing curves of the constructed antibody molecules. In experiment B, the effector cells were hPBMCs, the target cells were Daudi, the E:T ratio was 10:1, and the incubation time was 24 hours. In the other experiments (A, CJ), the effector cells were Pri-T cells, the target cells varied, the E:T ratio was 4:1, and the incubation time was 24 hours. Data were analyzed using GraphPad Prism software. A curve was fitted with four parameters, with the target cell killing rate on the ordinate and drug concentration on the abscissa. EC was then calculated. 50 value.

[0236] Figure 8 shows the results of the T cell activation experiment. hPBMCs and NCI-H929 cells were seeded in 96-well cell culture plates and treated with IMP-2023-045. After 24 hours, the supernatant was removed, and flow cytometry was used to detect T cell activation indicators and calculate CD8. + T cells (A) or CD4 + The proportion of CD69-positive T cells (B) was analyzed using GraphPad Prism software. A 4-parameter fitted curve was plotted with T cell activation rate on the ordinate and drug concentration on the abscissa to calculate the EC50. 50 value.

[0237] Figure 9 shows the results of the IMP-2023-045-promoted T cell proliferation assay. Freshly isolated hPBMCs were co-incubated with NCI-H929, followed by incubation with serially diluted IMP-2023-045 for 3 days. Cells were co-stained with anti-CD4, anti-CD8, and anti-Ki67 flow cytometry antibodies, and the T cell proliferation-promoting activity of IMP-2023-045 was analyzed. Data were analyzed using GraphPad Prism software. A 4-parameter fitted curve was plotted with Ki-67 on the ordinate and drug concentration on the abscissa to calculate the EC50. 50 value.

[0238] Figure 10 shows the experimental results of dexamethasone inhibiting cytokine release. Freshly isolated human PBMCs and NCI-H929 cells were seeded at a ratio of 10:1 in 96-well plates and incubated with serially diluted IMP-2023-045 or IMP-2023-045 + different concentrations of Dex for 24 hours. The supernatant was collected, and LDH levels and cytokine content were measured. Data were analyzed using GraphPad Prism software. A 4-parameter fitted curve was plotted with the cell killing rate and cytokine secretion on the ordinate and drug concentration on the abscissa. EC50 was then calculated. 50 Values. A shows the effect on cell-killing activity; BE shows the effects on inhibiting the release of IL-6, IFN-γ, TNF-α, and IL-2, respectively.

[0239] Figure 11 shows the tumor growth curve in an in vivo pharmacodynamic study.

[0240] Figure 12 shows the molecular structures of the constructed bispecific and trispecific antibodies; where A shows the constructed CD3 x GPRC5D series molecules; B shows the constructed CD3 x GPRC5D x BCMA series molecules; C shows the constructed CD3 x BCMA molecule; and D shows the constructed CD3 x EpCAM molecule. Detailed Implementation

[0241] Through extensive and in-depth research, the inventors have unexpectedly obtained, for the first time, a group of multispecific binding proteins that simultaneously target multiple antigens. The multispecific binding proteins of this invention comprise antigen-binding domains that bind at least two of the antigens selected from CD3, GPRC5D, and BCMA, and are constructed based on the aforementioned antigen-binding domains in Fab or scFv form. The multispecific antigen-binding proteins of this invention can simultaneously bind multiple target antigens (e.g., simultaneously binding GPRC5D and CD3, simultaneously binding BCMA and CD3, or simultaneously binding GPRC5D, BCMA, and CD3), and can be used as T-cell adaptors to connect target cells and T cells, inducing T-cell activation and proliferation and promoting T-cell killing. Based on this, the present invention was completed.

[0242] the term

[0243] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0244] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0245] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0246] As used herein, the term "treatment" refers to a clinical intervention aimed at altering the natural processes of the treated individual or cells during the course of a clinical lesion. Desired therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. For example, an individual is successfully "treated" if one or more cancer-related symptoms are alleviated or eliminated, including but not limited to reducing the proliferation of cancer cells (or destroying cancer cells), reducing symptoms caused by the disease, increasing the quality of life of the person suffering from the disease, reducing the dosage of other drug treatments required to treat the disease, and / or prolonging the individual's survival.

[0247] As used herein, the term "effective amount" refers to the amount of a dose or drug that is effective in treating a disease or symptom in a subject. In the case of cancer, an effective amount of the dose may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow down, and preferably terminate) the infiltration of cancer cells into peripheral organs; inhibit (i.e., to some extent slow down, and preferably terminate) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more of the symptoms associated with cancer to some extent. As is understood in clinical settings, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Therefore, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to have been administered in an effective amount if, in combination with one or more other agents, a desired result is achieved or realized.

[0248] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0249] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0250] Antibody

[0251] As used herein, the terms “antibody” or “immunoglobulin” are used in the broadest sense and explicitly encompass monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity or function. As used herein, the terms “immunoglobulin” (Ig) and “antibody” are used interchangeably.

[0252] The terms “natural antibody,” “full-length antibody,” “intact antibody,” and “complete antibody” are used interchangeably herein to refer to antibodies presenting their generally complete form, rather than antibody fragments as defined below. The terms specifically refer to antibodies having a heavy chain containing an Fc region. Natural antibodies are typically heterotetraglycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, the number of which varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, while the variable domains of the light chain are aligned with the variable domains of the heavy chain. Specific amino acid residues are thought to form the interface between the light chain variable domain and the heavy chain variable domain.

[0253] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0254] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0255] The term "antigen-binding fragment" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on the lower chain region; (iii) Fd fragments consisting of VH and CH1 domains; and (iv) Fv fragments consisting of the VH and VL domains of a single arm of the antibody. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.

[0256] This invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity, such as Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains.

[0257] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation. An epitope can be a discontinuous three-dimensional spatial site on the antigen, recognized by the antibody or antigen-binding fragment of this invention.

[0258] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M or l0 -10 M or lower affinity (KD) binding.

[0259] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0260] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0261] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0262] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242.

[0263] As used herein, the term "heavy chain constant region (CH)" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge region (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.

[0264] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region includes at least one of a constant κ domain or a constant λ domain.

[0265] The term "scFv" or "single-chain antibody" refers to a small molecule antibody that exists in a VL-VH or VH-VL configuration, consisting of VH and VL domains with a linker region between the VH and VL domains.

[0266] The term "Fab" refers to a monovalent fragment composed of VL, VH, CL, and CH1 domains, which forms a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0267] The terms "multispecific antibody" and "multispecific antigen-binding protein" are used interchangeably and refer to molecules that can bind to multiple different types of epitopes simultaneously, which may be located on the same target cell or different target cells.

[0268] In one embodiment, the present invention provides a bispecific antibody (also known as a bispecific T-cell connector) that simultaneously targets GPRC5D and CD3. In one embodiment, the present invention provides a bispecific antibody that simultaneously targets BCMA and CD3. In another embodiment, the present invention provides a trispecific antibody (also known as a trispecific T-cell connector) that simultaneously targets GPRC5D, BCMA, and CD3. The advantage of the bispecific or trispecific antibodies of the present invention is their ability to redirect specific polyclonal immune cells (e.g., T cells and NK cells) to tumor cells to enhance tumor killing. The antibodies of the present invention comprise antigen-binding fragments from two or more (e.g., three) monoclonal antibodies and function based on their antigen-binding capabilities. In one specific embodiment, the bispecific antibody of the present invention comprises a Fab fragment from an anti-GPRC5D antibody and an scFv from an anti-CD3 antibody. In one specific embodiment, the bispecific antibody of the present invention comprises an scFv from an anti-BCMA antibody and a Fab fragment from a CD3 antibody. In one specific embodiment, the trispecific antibody of the present invention comprises a Fab fragment from an anti-GPRC5D antibody, an scFv from an anti-BCMA antibody, and an scFv from an anti-CD3 antibody.

[0269] Bispecific or trispecific antibodies can be produced through chemical cross-linking or hybridoma techniques. Alternatively, bispecific or trispecific antibody molecules can be produced through recombinant techniques, such as by linking one or more scFv molecules to Fab molecules via linker peptides. Linker peptides are between 4 and 15 amino acids in length. Linker peptides can consist of various amino acids, such as repeating units of GGGGS, GG, SGGGGS, etc.

[0270] Polynucleotides, vectors and host cells

[0271] This invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof. The polynucleotide of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence of the antibody of this invention or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a sequence having the same amino acid sequence as the polypeptide of this invention, but with a different coding region sequence.

[0272] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence. The term "polynucleotide encoding a polypeptide" can include a polynucleotide encoding the polypeptide, or it can include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0273] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0274] The DNA sequence of the antibody or its fragment of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening.

[0275] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0276] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0277] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0278] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0279] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293, CHO K1 GS KO, ​​and CHO(DHFR-) cells.

[0280] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0281] The resulting monoclonal or polyclonal antibodies can be identified using conventional methods. For example, the binding specificity of the antibody can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of the antibody can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0282] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0283] Antibody-drug conjugates (ADCs)

[0284] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0285] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0286] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0287] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0288] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0289] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0290] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0291] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0292] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0293] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0294] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0295] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0296] Pharmaceutical Composition

[0297] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or its fusion protein or its ADC, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous, arterial, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension. The pharmaceutical composition of the present invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with GPRC5D and / or BCMA expression. Preferably, the pharmaceutical composition of the present invention is used for the prevention and / or treatment of cancers or tumors expressing GPRC5D and / or BCMA.

[0298] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0299] In one embodiment of the present invention, the multispecific antibody of the present invention can be used in combination with glucocorticoids to inhibit cytokine release without affecting the killing activity of target cells. Therefore, the pharmaceutical composition of the present invention may comprise the multispecific antibody of the present invention and a glucocorticoid (such as dexamethasone).

[0300] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0301] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by employing experiments not exceeding the norm. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0302] The main advantages of this invention include:

[0303] The multispecific antigen-binding protein of this invention can simultaneously bind to at least two antigens among GPRC5D, CD3, and BCMA, preventing antigen escape from a single target and improving therapeutic efficacy. When the multispecific antigen-binding protein simultaneously binds to CD3, as well as GPRC5D and / or BCMA, it can act as a T-cell connector, thereby linking T cells and target cells and promoting the killing effect of T cells on target cells. Furthermore, the multispecific antigen-binding protein of this invention also exhibits good safety, making it suitable for clinical applications.

[0304] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0305] Example 1: Construction of TriTE-Ab molecule

[0306] 1.1 TriTE-Ab Molecular Cloning

[0307] The construction process and methods for expression plasmids of bispecific and trispecific antibodies are similar. The following example of trispecific antibody construction will be used to illustrate the plasmid construction process.

[0308] Trispecific antibody molecules contain antibody sequences that bind to three different antigen targets. These three antibody sequences were amplified using overlap PCR to obtain the first polypeptide chain gene (heavy chain) and the second polypeptide chain gene (light chain), each containing its own Kozak sequence and signal peptide sequence. The DNA fragments encoding the heavy and light chains of the trispecific antigen-binding protein were double-digested with restriction endonucleases (EcoRI and NotI) and cloned into transient expression vectors to obtain transient expression vectors expressing the heavy and light chains (Figure 1, Figure 2). These transient expression vectors also contain an ampicillin resistance gene and a strong promoter CMV gene, which can promote high-level expression of the target gene in eukaryotic cells. Colonies were picked and sequenced; sequence alignment showed consistency with the theoretical sequence.

[0309] Positive clone plasmids were transformed into competent DH5α cells. Clones were selected and inoculated into 50 mL of E. coli culture medium and cultured overnight. The next day, bacterial cells were collected, and transfection-grade plasmids were extracted. Extraction of transfection-grade plasmids was performed according to the Plamid Plus Midi Kit (100) (QIAGEN, Cat No. 12945) instruction manual.

[0310] 1.2 TriTE-Ab protein expression

[0311] Transient expression was performed using CHO-S cells, which were cultured and expanded on ExpiCHO expression medium (Gibco, Cat No. A29100). The cell density was adjusted to 6 × 10⁶ cells / year according to the ExpiFectamine CHO transfection kit (Gibco, Cat No. A29129) manufacturer's instructions. 6 For cell / mL transfection, 20 μg of heavy chain plasmid and 20 μg of light chain plasmid were co-transfected into CHO-S cells at a 1:1 (mass ratio), and the cells were cultured in a CO2 incubator for 5 days. For transient transfection of CHO-S cells in a 50 mL system, the specific steps are as follows:

[0312] On day 1, prepare 50 mL of cells at a density of 6 × 10⁻⁶. 6 Cell slurry of cells / mL; add 20 μg heavy chain plasmid and 20 μg light chain plasmid to 2.0 mL OptiPRO. TM SFM, simultaneously with 160 μL Expi Fectamine TM Add CHO Reagent to 1.84 mL OptiPRO TM SFM; Plasmid solution with ExpiFectamineTM Mix the CHO Reagent solution and incubate at room temperature. Within 5 minutes, add 4 mL of the mixture to the cell culture medium. Transfer the cells to a 37°C, 5% CO2 incubator and shake at 110 rpm for further culture.

[0313] The next day, add 300 μL of Enhancer and 8 mL of ExpiCHO. TM Feed the cells into the cell shaker flask. Transfer the cells to a 37°C, 5% CO2 incubator and shake at 110 rpm for further culture.

[0314] On the fifth day, the supernatant was collected by centrifugation and filtered through a 0.22 μM filter.

[0315] 1.3 TriTE-Ab Molecular Purification

[0316] The target protein was purified using an IgG-CH1 affinity chromatography column (Thermo Fisher Scientific). The IgG-CH1 affinity chromatography column was equilibrated with phosphate-buffered saline (10 mM sodium phosphate buffer + 50 mM sodium chloride, pH 7.2), and cell culture supernatant was loaded. The column was then washed with phosphate-buffered saline (10 mM sodium phosphate buffer + 50 mM sodium chloride, pH 7.2), and the target protein was collected by elution with 50 mM NaAc buffer (pH 3.5). The pH of the eluent was adjusted to 4.5-5.5 using Tris buffer.

[0317] The constructed TriTE-Ab molecules are shown in Figure 12. In the figure, A represents the structures of CD3xGPRC5D bispecific antibody molecules IMP-2023-041, IMP-2023-042, and IMP-2023-043; B represents the structures of CD3xGPRC5DxBCMA trispecific molecules IMP-2023-045, IMP-2023-046, and IMP-2023-047; C represents the structure of CD3xBCMA bispecific molecule IMC-041; and D represents the structure of CD3xEpCAM bispecific molecule IMC-042.

[0318] Example 2: Detection of target protein expression in target protein-expressing cells

[0319] 2.1 Experimental Methods

[0320] The target protein expression cells used in the embodiments of this invention include NCI-H929, 293F-GPRC5D, Daudi, K562-CD20, K562-BCMA, hPBMCs, U266B1, KMS-26, MM.1S, and RPMI-8226. Among them, 293F-GPRC5D cells are adherent cells, MM.1S cells are semi-adherent and semi-suspended cells, and the other cells are suspension cells. The culture medium used for 293F-GPRC5D cells is DMEM (Life Technologies) containing 10% fetal bovine serum (FBS). The culture medium used for U266B1 cells is RPMI-1640 (Life Technologies) containing 20% ​​FBS, and the culture medium used for the other cells is RPMI-1640 containing 10% FBS.

[0321] The three cell lines 293F-GPRC5D, K562-CD20, and K562-BCMA were artificially constructed and stably expressed GPRC5D, CD20, or BCMA in 293F and K562 cells, respectively.

[0322] Prepare the cells to be tested at the same concentration, approximately 0.5-1 × 10⁻⁶. 6 Cells / mL: Take 50-100 μL, add APC anti-GPRC5D antibody (R&D Systems, FAB6300A) or PE anti-BCMA antibody (Biolegend, 357504), and incubate at room temperature in the dark. After 30-60 minutes, analyze using flow cytometry to calculate the mean fluorescence intensity (MFI) for each cell line.

[0323] 2.2 Experimental Results

[0324] Specific values ​​are shown in Table 3. The results indicate that, except for K562 and 293F-GPRC5D, other cell types such as RPMI-8226, KMS-26, MM.1S, NCI-H929, K562-BCMA, U266B1, and Daudi all express BCMA. 293F-GPRC5D only expresses GPRC5D. Except for NCI-H929, where GPRC5D expression was detectable, the GPRC5D levels in other cell types were near the detection background. K562 and K562-CD20, however, did not express either GPRC5D or BCMA.

[0325] Example 3: TriTE-Ab cell binding assay

[0326] T cell preparation: Human peripheral blood mononuclear cells (hPBMCs) (purchased from OriBiotech) were activated for 3 days with 5 μg / mL CD3 antibody (eBioscience, 16-0037-85) and 1 μg / mL CD28 antibody (eBioscience, 16-0289-85), followed by co-incubation with 10 ng / mL IL-2 (PeproTech, 200-02) for 3 days. Cells were then collected and cryopreserved. When T cells were needed for experiments, the cells were resuscitated and cultured with 10 ng / mL IL-2 for 1-3 days, and then collected for later use. The culture medium used was RPMI-1640 (Life Technologies) containing 10% fetal bovine serum (Life Technologies), referred to as complete medium.

[0327] The 293F-GPRC5D stable cell line expressed human GPRC5D in complete culture medium (10% fetal bovine serum + DMEM). NCI-H929 and K562-BCMA were both suspension cells in complete culture medium (10% fetal bovine serum + RPMI-1640). NCI-H929 cells simultaneously expressed BCMA and GPRC5D; K562-BCMA cells expressed BCMA.

[0328] First, dilute the cells to be used in PBS (flow cytometry buffer) containing 1% fetal bovine serum, then add 45 μL per tube at a rate of 1 × 10⁻⁶. 5 One cell line was seeded into an EP tube, and 5 μL of serially diluted test molecules were added. The mixture was incubated at room temperature. After 60 minutes, the cells were washed and resuspended in 50 μL of flow cytometry buffer, and PE-anti-human λ light chain antibody (BD Pharmaceuticals, 555797) was added. The cells were incubated at room temperature in the dark. After 45–60 minutes, the cells were analyzed using flow cytometry.

[0329] Because the analyte contains a human λ light chain, λ(+) positive cells represent cells that have bound the analyte. Data were analyzed using GraphPad Prism software, with cell binding rate on the ordinate and drug concentration on the x-axis. A 4-parameter fitted curve was plotted, and EC50 was calculated. 50 value.

[0330] The results showed that bispecific antibodies from different GPRC5D antibody clones bound to the 293F-Human GPRC5D stable cell line in a concentration-dependent manner (Figure 3).

[0331] IMP-2023-045 binds to T cells in EC 50 The value is 6.6 × 10-9 M, EC binding to 293F-GPRC5D (GPRC5D+) cells 50 The value is 5.2 × 10 -9 M, EC binding to K562-BCMA cells (BCMA+) and NCI-H929 cells (BCMA+GPRC5D+) 50 The values ​​are respectively 2.8 × 10 -7 M, 6.7 × 10 -9 M (Figure 4, Table 1).

[0332] Table 1. EC50 (M) values ​​of IMP-2023-045 binding to cells

[0333] Example 4: Cell killing experiment

[0334] 4.1 Experimental Methods

[0335] The target cells used in this experiment are the same as those used for protein expression detection.

[0336] The effector cells are T cells, and the specific preparations are as before.

[0337] Before the experiment, target cells were washed and centrifuged, stained with CFSE (Carboxyfluoresceinsuccinimidyl ester) (eBioscience, 65-0850-85) at room temperature for 10 minutes, then the staining was stopped, washed, and resuspended in complete culture medium. For effector cells (T cells), the cells were seeded at a ratio of effector cells to target cells of 4:1 (cell number); for effector cells (hPBMCs), the cells were seeded at a ratio of effector cells to target cells of 10:1. The target molecules were then incubated.

[0338] 24 hours later, 7-AAD (BD Pharmaceutical, 420404) was added to the corresponding wells of the 96-well plate. After staining for 10 minutes, the cells were analyzed by flow cytometry.

[0339] CFSE + Representing target cells, 7-AAD - Representing surviving cells, CFSE + 7-AAD - This represents surviving target cells. Cytotoxicity can be calculated using the following formula:

[0340] Data were analyzed using GraphPad Prism software. A 4-parameter fitted curve was plotted with cell killing rate on the ordinate and drug concentration on the x-axis. EC50 was then calculated. 50 value.

[0341] 4.2 Results of IMP-2023 series molecular cell killing experiments

[0342] Figure 5 shows the cell-killing results of various molecules when the effector cells are T cells. In Figure 5, the target cells of A are NCI-H929 cells that express both BCMA and GPRC5D. The CD3xGPRC5D bispecific antibody molecules IMP-2023-041 and IMP-2023-042 kill EC cells in these cells. 50 The concentrations were 4.0 and 7.2 pM, respectively, indicating that the bispecific antibody molecule can kill target cells through the GPRC5D antibody.

[0343] In Figure 5, target cells B are 293F-GPRC5D cells stably transfected with GPRC5D. The CD3xGPRC5D bispecific antibody molecules IMP-2023-041 and IMP-2023-042 kill EC cells in these cells. 50 The values ​​were 0.275 and 0.393 pM, respectively, and the trend of these results was the same as in Figure 5A. This indicates that the bispecific antibody molecule can kill target cells through the GPRC5D antibody.

[0344] Figure 6 shows the cell-killing results of various molecules when the effector cells are hPBMCs. In Figure 6, the target cells in A are NCI-H929 cells that double-express BCMA and GPRC5D. The trispecific antibody molecules IMP-2023-045 and IMP-2023-046 kill ECs in these cells. 50 The bispecific molecules IMP-2023-041 (CD3 xGPRC5D) and IMC-041 (CD3 x BCMA) were 5.3 and 9.8 pM, respectively, and were effective in killing EC2 cells. 50 The activity was between 6.2 and 13 pM, while the negative molecule IMC-042 (CD3 x EpCAM) had no effect, indicating that the trispecific antibody molecules can exert cell-killing effects through GPRC5D and BCMA antibodies.

[0345] In Figure 6, target cells B are GPRC5D monoexpressing 293F-GPRC5D cells, which are killed by the bispecific molecule IMP-2023-041 (CD3 x GPRC5D) in EC. 50 The EC50 value was 4.5 pM, while that of the negative molecule IMC-041 (CD3 xBCMA) was 4.5 pM. 50 The concentration was greater than 1000 pM, indicating that IMP-2023-041 (CD3 x GPRC5D) can specifically exert its cytotoxic effect through GPRC5D antibody. Further comparison of trispecific antibody molecules revealed that IMP-2023-045 and IMP-2023-046 had comparable activity in killing EC14 cells. 50 Both are 32pM.

[0346] Table 2 Summary of IMP-2023 Series Molecular Cell Killing

[0347] In summary, IMP-2023-045 and IMP-2023-046 effectively kill ECs in target cells expressing GPRC5D. 50 Both were at 32 pM, indicating that cells expressing only 293F-GPRC5D were easily killed by IMP-2023-045 and IMP-2023-046. The EC50 values ​​of IMP-2023-045 and IMP-2023-046 against target cells expressing both BCMA and GPRC5D were 5.3 pM and 9.8 pM, respectively, indicating that cells expressing both GPRC5D and BCMA were easily killed by IMP-2023-045 and IMP-2023-046.

[0348] 4.3 Results of IMP-2023-045 target cell killing assay

[0349] In Figure 7, the target cells of AD are cells expressing BCMA but not GPRC5D. IMP-2023-045 kills ECs in these cells. 50 Between 11 and 34 pM, the bispecific molecule IMP-2023-041 (CD3 x GPRC5D) did not kill target cells, indicating that IMP-2023-045 can specifically exert its effects through anti-BCMA rather than anti-GPRC5D.

[0350] In Figure 7, the target cells of E are 293F-GPRC5D cells that express GPRC5D but do not express BCMA. IMP-2023-045 kills EC cells in these cells. 50 At 11 pM, the bispecific molecule IMC-041 (CD3 x BCMA) did not kill target cells, indicating that IMP-2023-045 can exert its effects through anti-GPRC5D rather than anti-BCMA.

[0351] In Figure 7, the target cells of J are K562-CD20 cells that do not express either BCMA or GPRC5D, but are stably transfected with human CD20. IMP-2023-045 has no killing activity against them, while the positive molecule IMP-2023-026 (anti-CD3 x anti-CD19 x anti-CD20) has killing activity against them. This indicates that IMP-2023-045 has no activity against cells that do not express the target protein, demonstrating good selectivity.

[0352] In Figure 7, the target cells of FI are myeloma cells that express both BCMA and GPRC5D. IMP-2023-045 kills EC2 cells in these target cells.50 The effective concentration ranged from 1.2 to 5.3 pM, and its killing effect was essentially at the same level as that of the bispecific molecules IMP-2023-041 (CD3 x GPRC5D) and IMC-041 (CD3 x BCMA). However, the negative molecules IMC-042 (CD3 x EpCAM) and IMC-043 (CD3 x Her2) did not kill target cells, indicating that IMP-2023-045 can exert its effect through anti-GPRC5D or anti-BCMA.

[0353] Table 3: Summary of IMP-2023-045 molecular target cell killing and target protein expression table

[0354] In summary, IMP-2023-045 kills ECs in target cells expressing BCMA, GPRC5D, and both molecules. 50 The concentrations of 11-34 pM, 11-32 pM, and 1.2-5.3 pM, respectively, indicate that target cells expressing both BCMA and GPRC5D are more sensitive to IMP-2023-045.

[0355] Example 5: T cell activation experiment

[0356] 5.1 Experimental Methods

[0357] hPBMCs and NCI-H929 cells were resuspended in complete culture medium and seeded in 96-well cell culture plates at a ratio of 10:1. The detection antibodies were diluted to different concentrations with complete culture medium, and the test antibody or diluent was added to the corresponding well. The 96-well cell culture plates containing cells were then incubated in a CO2 incubator at 37°C. After 24 hours, APC-anti-CD4 antibody (Biolegend, 344614), PE-anti-CD8 antibody (Biolegend, 301008), and FITC-anti-CD69 antibody (Biolegend, 310904) were added, and the cells were incubated at room temperature for 30-60 minutes. Flow cytometry was used to analyze the cells and calculate CD4 counts. + T or CD8 + The proportion of CD69-positive cells within the T subset.

[0358] 5.2 Experimental Results

[0359] CD69 is a marker molecule for T cell activation. This experiment aims to determine whether IMP-2023-045 mediates T cell activation by detecting CD69 expression in T cells.

[0360] The data obtained from the flow cytometer were subjected to four-parameter equation fitting analysis using Graphpad Prism software to obtain the dose-response curve and the half-maximum effective concentration (EC50).50 As shown in Figure 8, IMP-2023-045 can induce CD8 in a concentration-dependent manner. + T cells or CD4 + CD69 expression on T cells, its EC 50 They are 35pM and 42pM respectively.

[0361] Conclusion: IMP-2023-045 can induce T cell activation in human PBMCs in a concentration-dependent manner.

[0362] Example 6: T cell proliferation experiment

[0363] 6.1 Experimental Methods

[0364] hPBMCs and NCI-H929, resuspended in complete culture medium, were seeded at a ratio of 5:1 in 96-well cell culture plates. Detection antibodies were diluted to different concentrations with complete culture medium, and either the test antibody or diluent was added to the corresponding well. Finally, the 96-well cell culture plates containing cells were placed in a CO2 incubator and incubated at 37°C.

[0365] 72 hours later, T cells were collected and treated using the Fixation / Permeabilization Solution Kit (Invitrogen, 00-5523-00), and APC-anti-CD4 antibody (Biolegend, 344614), PE-anti-CD8 antibody (Biolegend, 301008), and FITC-anti-Ki67 antibody (BD Pharmingen, 556026) were added. The cells were then incubated at room temperature in the dark for 45–60 minutes. Finally, flow cytometry was used to analyze the cells and calculate the CD4+ content within the T cell population. + Ki67 + or CD8 + Ki67 + Proportion.

[0366] 6.2 Experimental Results

[0367] Ki67 is a marker protein for cell proliferation. This experiment investigated the activity of IMP-2023-045 in inducing T cell proliferation by detecting Ki67 expression in T cells.

[0368] The results showed that, under the same conditions, CD4 + T cell proliferation rate is less than CD8 + T cell proliferation rate, its EC 50 The values ​​were 26 and 8.9 pM, respectively, as shown in Figure 9.

[0369] Conclusion: The results show that IMP-2023-045 can induce T cell proliferation in a concentration-dependent manner.

[0370] Example 7: Dexamethasone inhibits cytokine release

[0371] 7.1 Experimental Methods

[0372] hPBMCs and NCI-H929 cells were resuspended in complete culture medium and seeded at an effector cell:target cell ratio of 10:1. Cells were incubated with IMP-2023-045 or IMP-2023-045 + different concentrations of dexamethasone (Dex) (Sigma, 50-02-2). After 24 hours, the cell supernatant or cell lysate was collected for the following experiments.

[0373] In this experiment, an LDH (lactate dehydrogenase) kit (Promega, batch number: G1782) was used to determine the killing curve of IMP-2023-045-mediated T cells against NCI-H929 cells with and without Dex, according to the method provided in the manufacturer's instructions. The specific method was as follows: cell supernatant or cell lysis buffer was taken, LDH substrate was added, the reaction was terminated after 30 minutes, and the absorbance value (OD490) was measured at 490 nm. During cell death, LDH is released into the culture medium; therefore, the LDH content in the cell supernatant is related to the degree of cell death. The cell death rate was calculated using the following formula:

[0374] in,

[0375] [OD490] 样品 Absorbance values ​​derived from the supernatant of wells containing NCI-H929 cells, hPBMCs, and IMP-2023-045

[0376] [OD490] 最小 Absorbance values ​​derived from the supernatant of wells containing NCI-H929 cells and hPBMCs

[0377] [OD490] 最大 The absorbance value originates from the mixed liquid in the wells of the cell lysis buffer added during NCI-H929 cell detection.

[0378] [OD490] 本底 Absorbance value derived from NCI-H929 cell culture medium

[0379] Using the same sample, relevant cytokines were detected according to the instructions of the IL-2 ELISA kit (R&D, DY202), IL-6 ELISA kit (R&D, DY206), TNF-α ELISA kit (R&D, DY210), and IFN-γ ELISA kit (R&D, DY285B).

[0380] 7.2 Experimental Results

[0381] T cell activation and proliferation can cause cytokine release syndrome (CRS), and fulminant CRS can be life-threatening. Glucocorticoids, such as dexamethasone (Dex), can effectively alleviate CRS caused by T cell activation, and are therefore a commonly used clinical treatment for controlling CRS.

[0382] The results are shown in Figure 10 and Table 4 below. The results indicate that without Dex, or with 3×10... -7 M or 3×10 -6 M Dex, IMP-2023-045 mediates the killing activity of T cells against NCI-H929 cells. 50 The concentrations were 11, 26, and 28 pM, respectively (Figure 10, A). This means that Dex had almost no effect on the cytotoxic activity of IMP-2023-045, but almost completely inhibited the release of IL-6 (Figure 10, B), and inhibited the secretion of IFNγ, TNF-α, and IL-2 to varying degrees (Figure 10, C, D, and E).

[0383] Table 4. Dexamethasone inhibits cytokine release.

[0384] Conclusion: Dexamethasone can inhibit cytokine release without affecting the killing activity of IMP-2023-045.

[0385] Example 8: In vivo antitumor pharmacodynamics study

[0386] 8.1 Experimental Methods

[0387] Collect 293-GPRC5D cells (obtained by transfecting 293 cells with human GPRC5D) in the logarithmic growth phase from in vitro culture, resuspend them in PBS, and adjust the cell density to 5 × 10⁶ cells / year. 7 Cells / mL, 5 × 10⁶ per mouse 6 0.1 mL of cells was injected subcutaneously into each mouse. The day of vaccination was defined as Day 0.

[0388] Two days later (Day 2), human PBMCs were resuspended in PBS, and the cell density was adjusted to 2.5 × 10⁻⁶. 7 Cells / mL, 5 × 10⁶ per mouse 6 Immunological reconstitution was performed by intraperitoneal injection of 0.2 mL of cells per mouse.

[0389] On day 10 post-inoculation, when the tumor had grown to an average volume of approximately 120 mm... 3Mice with tumors conforming to a normal distribution were then randomly divided into four groups: a solvent control group (PBS), a low-dose subcutaneous IMP-2023-045 group (IMP-2023-045, 100 μg / kg, SC), a high-dose subcutaneous IMP-2023-045 group (IMP-2023-045, 300 μg / kg, SC), and an intraperitoneal IMP-2023-045 group (IMP-2023-045, 300 μg / kg, IP). Animals in each group received the medication subcutaneously or intraperitoneally, once every other day, for a total of 3 weeks. Afterward, drug administration was discontinued, and the study concluded on Day 34.

[0390] The long axis (L) and short axis (W) of tumors in tumor-bearing mice were measured using calipers 2-3 times per week, and the tumor volume (TV) and tumor growth inhibition rate (TGI%) were calculated according to the following formulas. Where avT0 and avT... i The mean tumor volumes (avC0 and avC) of the treatment group mice at the time of grouping (Day 7) and Day 7+i are respectively. i The values ​​represent the average tumor volume of the solvent control mice at the time of grouping (Day 7) and Day 7+i, respectively. Tumor volume (TV) = (L × W) 2 ) / 2

[0391] 8.2 Experimental Results

[0392] The results are shown in Figure 11. The results indicated that subcutaneous injection of IMP-2023-045 exhibited dose-dependent antitumor efficacy in 293-GPRC5D tumor-bearing mice with human PBMC immune reconstitution. The TGI% of the low-dose subcutaneous IMP-2023-045 group (100 μg / kg, SC) and the high-dose subcutaneous IMP-2023-045 group (300 μg / kg, SC) on Day 34 were 58.2% and 92.4%, respectively. Compared with the solvent control group at the same time point, the P values ​​were 0.0241 and 0.0081, respectively, which were statistically significant.

[0393] At the same dose (300 μg / kg), the antitumor effects of subcutaneous and intraperitoneal injection of IMP-2023-045 were similar. The TGI% of the two groups on Day 34 were 92.4% and 76.8%, respectively. Compared with the solvent control group at the same time point, the P values ​​were 0.0081 and 0.0082, respectively, which were statistically significant.

[0394] Conclusion: Subcutaneous administration of IMP-2023-045 can inhibit the growth of xenografts in GPRC5D-positive 293-GPRC5D mice in a dose-dependent manner, and the tumor-inhibiting effect is similar to that of intraperitoneal injection of the same dose.

[0395] Sequence information of the present invention

[0396] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A multispecific antigen-binding protein, characterized in that, The antigen-binding protein comprises at least two antigen-binding domains selected from the group consisting of: (Z1) one or more CD3-binding domains, (Z2) one or more GPRC5D-binding domains, and (Z3) one or more BCMA-binding domains.

2. The multispecific antigen-binding protein as described in claim 1, characterized in that, The antigen-binding domain is in the form of a Fab fragment, a single-chain Fv (scFv), an F(ab')2 fragment, a single-domain antibody (sdAb), or a combination thereof.

3. The multispecific antigen-binding protein as described in claim 1, characterized in that, The antigen-binding domain that binds to GPRC5D includes the following three heavy chain variable regions (CDRs): HCDR1 has an amino acid sequence as shown in SEQ ID NO:22 or 25; HCDR2, which has the amino acid sequence shown in SEQ ID NO:23; and HCDR3 has an amino acid sequence as shown in SEQ ID NO:24 or 26; And the following three light chain variable regions (CDRs): LCDR1 has the amino acid sequence shown in SEQ ID NO:27; LCDR2, which has the amino acid sequence shown in SEQ ID NO:28; and LCDR3 has the amino acid sequence shown in SEQ ID NO:

29.

4. The multispecific antigen-binding protein as described in claim 3, characterized in that, The antigen-binding domain that binds to GPRC5D includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, or 3; and / or The light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

5. The multispecific antigen-binding protein as described in claim 3 or 4, characterized in that, The antigen-binding domain that binds to GPRC5D is selected from the group consisting of scFv, Fab, or a combination thereof.

6. The multispecific antigen-binding protein as described in claim 5, characterized in that, The antigen-binding domain that binds to GPRC5D is Fab, which includes a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:1, 2, or 3, and a light chain variable region of an amino acid sequence as shown in SEQ ID NO:

4. And the heavy chain constant region CH1 of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region CL of the amino acid sequence shown in SEQ ID NO:

10.

7. The multispecific antigen-binding protein as described in claim 1, characterized in that, The antigen-binding domain that binds to BCMA includes the following three heavy chain variable regions (CDRs): HCDR1 has the amino acid sequence shown in SEQ ID NO:30; HCDR2, which has the amino acid sequence shown in SEQ ID NO:31; and HCDR3 has the amino acid sequence shown in SEQ ID NO:32; And the following three light chain variable regions (CDRs): LCDR1 has the amino acid sequence shown in SEQ ID NO:33; LCDR2, which has the amino acid sequence shown in SEQ ID NO:34; and LCDR3 has the amino acid sequence shown in SEQ ID NO:

35.

8. The multispecific antigen-binding protein as described in claim 7, characterized in that, The antigen-binding domain that binds to BCMA includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7; and / or The light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

9. The multispecific antigen-binding protein as described in claim 7 or 8, characterized in that, The antigen-binding domain that binds to BCMA is selected from the group consisting of scFv, Fab, or a combination thereof.

10. The multispecific antigen-binding protein as described in claim 1, characterized in that, The antigen-binding domain that binds to CD3 includes the following three heavy chain variable regions (CDRs): HCDR1 has the amino acid sequence shown in SEQ ID NO:44; HCDR2, which has the amino acid sequence shown in SEQ ID NO:45; and HCDR3 has the amino acid sequence shown in SEQ ID NO:46; And the following three light chain variable regions (CDRs): LCDR1 has the amino acid sequence shown in SEQ ID NO:47; LCDR2, which has the amino acid sequence shown in SEQ ID NO:48; and LCDR3 has the amino acid sequence shown in SEQ ID NO:

49.

11. The multispecific antigen-binding protein as described in claim 10, characterized in that, The antigen-binding domain that binds to CD3 includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5, and / or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

6.

12. The multispecific antigen-binding protein as described in claim 10, characterized in that, The antigen-binding domain that binds to CD3 is selected from the group consisting of scFv, Fab, or a combination thereof.

13. The multispecific antigen-binding protein as described in claim 12, characterized in that, The antigen-binding domain that binds to CD3 is Fab, which includes a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:5 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:6, a heavy chain constant region CH1 of the amino acid sequence shown in SEQ ID NO:9, and a light chain constant region CL of the amino acid sequence shown in SEQ ID NO:

36.

14. The multispecific antigen-binding protein as described in claim 1, characterized in that, The multispecific antigen-binding protein is either a bispecific antigen-binding protein or a trispecific antigen-binding protein.

15. The multispecific antigen-binding protein as described in claim 14, characterized in that, The bispecific antigen-binding protein comprises one or more antigen-binding domains that bind GPRC5D and one or more antigen-binding domains that bind CD3; or The bispecific antigen-binding protein includes one or more antigen-binding domains that bind BCMA and one or more antigen-binding domains that bind CD3. Wherein, the antigen-binding domain that binds GPRC5D is Fab or scFv, the antigen-binding domain that binds BCMA is Fab or scFv, and the antigen-binding domain that binds CD3 is Fab or scFv.

16. The multispecific antigen-binding protein as described in claim 15, characterized in that, The bispecific antigen-binding protein comprises an antigen-binding domain for binding GPRC5D and an antigen-binding domain for binding CD3; wherein the antigen-binding domain for binding GPRC5D is Fab, and the antigen-binding domain for binding CD3 is scFv; or, the antigen-binding domain for binding GPRC5D is scFv, and the antigen-binding domain for binding CD3 is Fab; or The bispecific antigen-binding protein comprises an antigen-binding domain that binds to BCMA and an antigen-binding domain that binds to CD3; wherein the antigen-binding domain that binds to BCMA is scFv and the antigen-binding domain that binds to CD3 is Fab; or, the antigen-binding domain that binds to BCMA is Fab and the antigen-binding domain that binds to CD3 is scFv.

17. The multispecific antigen-binding protein as described in claim 14, characterized in that, The trispecific antigen-binding protein includes one or more antigen-binding domains that bind GPRC5D, one or more antigen-binding domains that bind CD3, and one or more antigen-binding domains that bind BCMA. Wherein, the antigen-binding domain that binds GPRC5D is Fab or scFv, the antigen-binding domain that binds BCMA is Fab or scFv, and the antigen-binding domain that binds CD3 is Fab or scFv.

18. The multispecific antigen-binding protein as described in claim 17, characterized in that, The trispecific antigen-binding protein includes an antigen-binding domain that binds GPRC5D, an antigen-binding domain that binds CD3, and an antigen-binding domain that binds BCMA. Wherein, the antigen-binding domain binding GPRC5D is Fab, the antigen-binding domain binding CD3 is scFv, and the antigen-binding domain binding BCMA is scFv; or The antigen-binding domain for GPRC5D is scFv, the antigen-binding domain for CD3 is Fab, and the antigen-binding domain for BCMA is scFv; or The antigen-binding domain for GPRC5D is scFv, the antigen-binding domain for CD3 is scFv, and the antigen-binding domain for BCMA is Fab.

19. The multispecific antigen-binding protein according to any one of claims 15-18, characterized in that, The antigen-binding domain that binds to GPRC5D includes the following three heavy chain variable regions (CDRs): HCDR1 has an amino acid sequence as shown in SEQ ID NO:22 or 25; HCDR2, which has the amino acid sequence shown in SEQ ID NO:23; and HCDR3 has an amino acid sequence as shown in SEQ ID NO:24 or 26; And the following three light chain variable regions (CDRs): LCDR1 has the amino acid sequence shown in SEQ ID NO:27; LCDR2, which has the amino acid sequence shown in SEQ ID NO:28; and LCDR3 has the amino acid sequence shown in SEQ ID NO:29; The antigen-binding domain that binds to BCMA includes the following three heavy chain variable regions (CDRs): HCDR1 has the amino acid sequence shown in SEQ ID NO:30; HCDR2, which has the amino acid sequence shown in SEQ ID NO:31; and HCDR3 has the amino acid sequence shown in SEQ ID NO:32; And the following three light chain variable regions (CDRs): LCDR1 has the amino acid sequence shown in SEQ ID NO:33; LCDR2, which has the amino acid sequence shown in SEQ ID NO:34; and LCDR3 has the amino acid sequence shown in SEQ ID NO:35; The antigen-binding structure that binds to CD3 includes the following three heavy chain variable regions (CDRs): HCDR1 has the amino acid sequence shown in SEQ ID NO:44; HCDR2, which has the amino acid sequence shown in SEQ ID NO:45; and HCDR3 has the amino acid sequence shown in SEQ ID NO:46; And the following three light chain variable regions (CDRs): LCDR1 has the amino acid sequence shown in SEQ ID NO:47; LCDR2, which has the amino acid sequence shown in SEQ ID NO:48; and LCDR3 has the amino acid sequence shown in SEQ ID NO:

49.

20. The multispecific antigen-binding protein according to any one of claims 15-18, characterized in that, The antigen-binding domain that binds to GPRC5D includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:1, 2, or 3; and / or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

4. The antigen-binding domain that binds to BCMA includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7; and / or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:8; The antigen-binding domain that binds to CD3 includes a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:5, and / or a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

6.

21. The multispecific antigen-binding protein as described in claim 15, characterized in that, The heavy chain amino acid sequence of the bispecific binding protein is shown in SEQ ID NO:13, 15 or 16, and the light chain amino acid sequence is shown in SEQ ID NO:14; or The heavy chain amino acid sequence of the bispecific binding protein is shown in SEQ ID NO:18, and the light chain amino acid sequence is shown in SEQ ID NO:

19.

22. The multispecific antigen-binding protein as described in claim 17, characterized in that, The heavy chain amino acid sequence of the three specific binding protein is shown in SEQ ID NO:13, 15 or 16, and the light chain amino acid sequence is shown in SEQ ID NO:

17.

23. An anti-GPRC5D antibody or its antigen-binding fragment, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region has the following complementary determining regions (CDRs): HCDR1 as shown in SEQ ID NO:22 or 25 HCDR2 shown in SEQ ID NO:23, and HCDR3 as shown in SEQ ID NO:24 or 26; Furthermore, the light chain variable region has the following complementary determinant region (CDR): LCDR1 shown in SEQ ID NO:27 LCDR2 shown in SEQ ID NO:28, and LCDR3 as shown in SEQ ID NO:

29.

24. The anti-GPRC5D antibody or its antigen-binding fragment as described in claim 23, characterized in that, The heavy chain variable region of the antibody has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, or 3; and / or The light chain variable region of the antibody has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

25. An anti-BCMA antibody or its antigen-binding fragment, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region has the following complementary determining regions (CDRs): HCDR1, as shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, and HCDR3 as shown in SEQ ID NO:32; Furthermore, the light chain variable region has the following complementary determinant region (CDR): LCDR1 shown in SEQ ID NO:33 LCDR2 shown in SEQ ID NO:34, and LCDR3 as shown in SEQ ID NO:

35.

26. The anti-BCMA antibody or its antigen-binding fragment as described in claim 25, characterized in that, The heavy chain variable region of the antibody has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7; and / or The light chain variable region of the antibody contains a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

27. A recombinant protein, characterized in that, The recombinant protein comprises: (i) the multispecific antigen-binding protein of any one of claims 1-22, the anti-GPRC5D antibody of claim 23 or its antigen-binding fragment thereof, or the anti-BCMA antibody of claim 25 or its antigen-binding fragment thereof; and (ii) Tag sequences that assist in expression and / or purification.

28. A polynucleotide encoding a multispecific antigen-binding protein as claimed in any one of claims 1-22, an anti-GPRC5D antibody as claimed in claim 23, an anti-BCMA antibody as claimed in claim 25, or a recombinant protein as claimed in claim 27.

29. A vector comprising the polynucleotide as described in claim 28.

30. A genetically engineered host cell, said host cell containing the vector as described in claim 29, or having an exogenous polynucleotide as described in claim 28 integrated into its genome.

31. An antibody conjugate, characterized in that, The antibody conjugate contains: (a) Antibody portion, wherein the antibody portion is selected from the group consisting of: The multispecific antigen-binding protein as described in any one of claims 1-22, the anti-GPRC5D antibody as described in claim 23 or its antigen-binding fragment thereof, the anti-BCMA antibody as described in claim 25 or its antigen-binding fragment thereof, or the recombinant protein as described in claim 27; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of detectable markers, drugs, or combinations thereof.

32. Use of an active ingredient, said active ingredient being selected from the group consisting of: a multispecific antigen-binding protein as described in any one of claims 1-22, an anti-GPRC5D antibody or its antigen-binding fragment as described in claim 23, an anti-BCMA antibody or its antigen-binding fragment as described in claim 23, a recombinant protein as described in claim 27, or an antibody conjugate as described in claim 31, or a combination thereof; said active ingredient is used for: (i) To prepare medicines for the prevention and / or treatment of diseases associated with GPRC5D and / or BCMA expression; or (ii) Prepare reagents for detecting diseases associated with GPRC5D and / or BCMA expression.

33. The use as described in claim 32, characterized in that, The disease described is cancer or tumor.

34. The use as described in claim 33, characterized in that, The cancer or tumor mentioned is a solid tumor or a hematoma.

35. The use as described in claim 33, characterized in that, The cancer or tumor mentioned is multiple myeloma.

36. A pharmaceutical composition comprising (i) An active ingredient selected from the group consisting of: a multispecific antigen-binding protein as described in any one of claims 1-22, an anti-GPRC5D antibody as described in claim 23 or an antigen-binding fragment thereof, an anti-BCMA antibody as described in claim 25 or an antigen-binding fragment thereof, a recombinant protein as described in claim 27, or an antibody conjugate as described in claim 31, or a combination thereof; (ii) Pharmaceutically acceptable carriers.

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