Bispecific antibodies targeting G protein coupled receptors

Abstract

A bispecific antibody targeting a G protein coupled receptor is provided. Specifically provided are bispecific antibodies, nucleic acids encoding them, vectors comprising the nucleic acids, cells comprising the vectors, pharmaceutical compositions comprising the multispecific antibodies, and uses thereof in treating subjects with related diseases.

Description

[0001] This application is a divisional application of the invention patent application filed on April 26, 2024, with Chinese application number CN202480027332.0 and invention title "Bispecific antibody targeting G protein-coupled receptor".

[0002] Cross-references This application claims the benefit of Chinese Patent Application No. CN202310484445.0, filed on April 28, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to bispecific antibodies, and more particularly to bispecific antibodies targeting GPRC5D and CD3. This disclosure also relates to methods for preparing and using the said bispecific antibodies. Background Technology

[0004] Multiple myeloma (MM) is the second most common hematologic malignancy after non-Hodgkin's lymphoma. Its pathological condition is characterized by the abnormal proliferation of plasma cells in the bone marrow, which crowds out the development of normal blood cells, leading to a decrease in blood cells, bone damage, and kidney damage.

[0005] GPRC5D (G protein-coupled receptor class C group 5 member D) belongs to the retinoic acid-induced orphan G protein-coupled receptor (RAIG) family. It is a 7-transmembrane protein composed of 345 amino acid residues. Its normal physiological function is related to duracin structure, but its specific biological function or ligands remain unclear. Some studies have shown that GPRC5D has an expression threshold exceeding 50% in plasma cells derived from malignant bone marrow in 65% of multiple myeloma patients, and its expression is independent of BCMA (B cell maturation antigen). Other studies have shown that GPRC5D overexpression is associated with tumor burden and poor prognosis in multiple myeloma patients. In normal tissues, GPRC5D is not expressed or is expressed at very low levels, except in hair follicles of the skin and plasma cells derived from bone marrow. GPRC5D may provide new treatment options for multiple myeloma patients, highlighting its clinical value.

[0006] As a potential therapeutic target, several antibodies targeting GPRC5D are under development, but the options are still limited and more are needed. Invention Summary This disclosure provides a bispecific antibody targeting GPRC5D and CD3, as well as related nucleic acids, vectors, cells, pharmaceutical compositions, preparation methods, and uses that can encode the provided antibody.

[0007] In one aspect, this disclosure provides a bispecific antibody comprising a first antigen-binding portion that binds to GPRC5D and a second antigen-binding portion that binds to CD3, wherein the first antigen-binding portion comprises: (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (3) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 60, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 61, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (5) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14; (6) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22; (7) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30; or (8) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0008] In one aspect, this disclosure also provides a bispecific antibody comprising a first antigen-binding moiety binding to GPRC5D and a second antigen-binding moiety binding to CD3, wherein the first antigen-binding moiety comprises: (1) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8; (2) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 15, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 16; (3) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24; (4) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 31, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 32; (5) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 39, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 40; (6) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 62, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (7) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 63, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (8) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 64, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (9) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 65, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (10) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; or (11) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 69.

[0009] In some implementations, the first antigen-binding portion includes: (1) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 8; (2) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 16; (3) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 24; (4) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 32; (5) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 40; (6) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (7) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (8) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (9) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (10) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; or (11) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 69.

[0010] In some embodiments, the second antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 94, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 95, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 96, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 97, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 98, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 99.

[0011] In some embodiments, the second antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 100, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 101.

[0012] In some specific embodiments, the bispecific antibody comprises a first antigen-binding portion that binds to GPRC5D and a second antigen-binding portion that binds to CD3; wherein the first antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; the second antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 94, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 95, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 96, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 97, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 98, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 99.

[0013] In some other specific embodiments, the bispecific antibody comprises a first antigen-binding portion that binds to GPRC5D and a second antigen-binding portion that binds to CD3; wherein the first antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38; the second antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 94, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 95, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 96, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 97, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 98, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 99.

[0014] In some specific embodiments, the bispecific antibody comprises a first antigen-binding region that binds to GPRC5D and a second antigen-binding region that binds to CD3, wherein the first antigen-binding region comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; and the second antigen-binding region comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 100 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101.

[0015] In some other specific embodiments, the bispecific antibody comprises a first antigen-binding region that binds to GPRC5D and a second antigen-binding region that binds to CD3, wherein the first antigen-binding region comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69; and the second antigen-binding region comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 100 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101.

[0016] In some embodiments, the bispecific antibody of this disclosure comprises an Fc domain consisting of two Fc polypeptides.

[0017] In some embodiments, the first antigen-binding portion and the second antigen-binding portion are each independently Fab, scFv, scFab (single-chain Fab), or other structures. In some embodiments, the first antigen-binding portion is Fab, and the second antigen-binding portion is scFv.

[0018] In some embodiments, the bispecific antibody of this disclosure has a first antigen-binding moiety of Fab and a second antigen-binding moiety of scFv. The first antigen-binding moiety is fused at the C-terminus of its Fab heavy chain or Fab light chain to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding moiety is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain. In a more specific embodiment, the bispecific antibody consists of three polypeptide chains, wherein the first polypeptide chain contains the Fab light chain of the first antigen-binding moiety, the second polypeptide chain contains the Fab heavy chain of the first antigen-binding moiety and one of the Fc peptides in the Fc domain, and the third polypeptide chain contains the second antigen-binding moiety and another Fc peptide in the Fc domain.

[0019] In some implementations, the bispecific antibody is bivalent.

[0020] On the other hand, this disclosure provides an antibody or antigen-binding moiety thereof that binds to GPRC5D, comprising: (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (3) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 60, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 61, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (5) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14; (6) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22; (7) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30; or (8) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0021] In one aspect, this disclosure also provides an antibody or antigen-binding moiety thereof that binds to GPRC5D, comprising: (1) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8; (2) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 15, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 16; (3) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24; (4) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 31, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 32; (5) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 39, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 40; (6) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 62, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (7) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 63, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (8) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 64, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (9) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 65, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (10) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; or (11) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 69.

[0022] In some embodiments, the antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 62, 63, 64, 65, 66, or 68, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67 or 69.

[0023] In some embodiments, the antibody binding to GPRC5D or its antigen-binding portion comprises: (1) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (2) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (3) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (4) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (5) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 69; or (6) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 69.

[0024] In some embodiments, the antibody binding to GPRC5D or its antigen-binding portion comprises: (1) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (2) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (3) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (4) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; or (5) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69.

[0025] In one aspect, this disclosure provides isolated nucleic acids comprising nucleotide sequences encoding the bispecific antibodies or antibody- or antigen-binding portions thereof described in this disclosure.

[0026] In one respect, this disclosure provides a carrier containing the nucleic acid described herein.

[0027] In one aspect, this disclosure provides a host cell containing the nucleic acid or the vector described herein.

[0028] In one aspect, this disclosure provides antibody-drug conjugates comprising the antibody of this disclosure that binds to GPRC5D or its antigen-binding portion.

[0029] In one aspect, this disclosure provides an anti-GPRC5D chimeric antigen receptor comprising an antibody of this disclosure that binds to GPRC5D.

[0030] In one aspect, this disclosure provides engineered immune effector cells that contain the chimeric antigen receptor of this disclosure.

[0031] On the other hand, this disclosure provides a method for preparing the bispecific antibody or antibody or antigen-binding portion thereof as described herein, comprising culturing the host cells to express the bispecific antibody or antibody or antigen-binding portion thereof, and isolating and purifying the bispecific antibody or antibody or antigen-binding portion thereof in the system.

[0032] On the other hand, this disclosure provides pharmaceutical compositions comprising the antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immune effector cells or the bispecific antibody, and pharmaceutically acceptable excipients.

[0033] On the other hand, this disclosure provides the use of the antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immune effector cells, the bispecific antibody, or the pharmaceutical composition in the preparation of a medicament for treating diseases expressing GPRC5D.

[0034] On the other hand, this disclosure provides a method for treating a disease expressing GPRC5D, comprising administering to a subject in need the antibody or its antigen-binding portion thereof, the antibody-drug conjugate, the engineered immune effector cells, the bispecific antibody, or the pharmaceutical composition. Alternatively, this disclosure provides a method for treating a disease expressing GPRC5D, comprising administering to a subject in need a therapeutically effective amount of the antibody or its antigen-binding portion thereof, the antibody-drug conjugate, the engineered immune effector cells, the bispecific antibody, or the pharmaceutical composition.

[0035] The bispecific antibody targeting GPRC5D and CD3 disclosed herein exhibits good anti-tumor efficacy and / or safety. Attached Figure Description

[0036] Figure 1This refers to the partial detection of mouse anti-hGPRC5D antibody and CHO-K1-hGPRC5D by ELISA. hi OD bound to cells, CHO-K1-cynoGPRC5D cells, and CHO-K1-murineGPRC5D cells 450 nm ; Figure 2 MFI and EC were used to detect the binding of chimeric antibodies to cells expressing high levels of hGPRC5D in FACS. 50 ; Figure 3 MFI was used to detect the binding of chimeric antibodies to cells with low hGPRC5D expression in FACS. Figures 4A-4B For FACS detection of MFI of anti-GPRC5D antibody binding to different cells, among which Figure 4A For NCI-H929 cells, Figure 4B MFI that binds to MM.1S cells; Figures 5A-5B For FACS detection of MFI of anti-GPRC5D antibody binding to different cells, among which Figure 5A CHO-K1-cynoGPRC5D cells, Figure 5B CHO-K1-murine GPRC5D cells; Figures 6A-6C For FACS detection of MFI of anti-GPRC5D antibody binding to different cells, among which Figure 6A CHO-K1-GPRC5A cells, Figure 6B CHO-K1-GPRC5B cells, Figure 6C CHO-K1-GPRC5C cells; Figure 7 MFI for FACS detection of anti-GPRC5D antibody binding to CHO-K1 cells; Figure 8 MFI was used to detect the binding of chimeric and humanized anti-GPRC5D antibodies to NCI-H929 cells in FACS. Figure 9 This is a schematic diagram of the structure of an exemplary bispecific antibody of this disclosure; Figures 10A-10C For FACS detection of MFI by anti-GPRC5D / anti-CD3 bispecific antibody binding to different cells, among which Figure 10A For RPMI-8226 cells, Figure 10B For MM.1S cells, Figure 10C NCI-H929 cells; Figure 11 For FACS detection of anti-GPRC5D / anti-CD3 bispecific antibody and CD3+ T-cell-bound MFI; Figures 12A-12C The lysis rate of different cells by the anti-GPRC5D / anti-CD3 bispecific antibody, among which Figure 12A For NCI-H929 cells, Figure 12B For MM.1S cells, Figure 12C RPMI-8226 cells; Figures 13A-13D The effect of anti-GPRC5D / anti-CD3 bispecific antibody on T cell activation in the presence of target cells MM.1S was investigated. CD69 and CD25 are markers of T cell activation. Figure 13A CD25 + Cell number as a percentage of CD4 + The proportion of cell number Figure 13B CD69 + Cell number as a percentage of CD4 + The proportion of cell number Figure 13C CD25 + Cell number as a percentage of CD8 + The proportion of cell number Figure 13D CD69 + Cell count as a percentage of CD8 + The proportion of cell numbers; Figures 14A-14D The study investigated the release levels of different cytokines after co-incubating different concentrations of anti-GPRC5D / anti-CD3 bispecific antibodies with target cells MM.1S and effector cells PBMC for 24 h. Figure 14A For IL-2, Figure 14B For IL-6, Figure 14C It is TNF-α, Figure 14D For IFN-γ; Figure 15 To investigate the effect of anti-GPRC5D / anti-CD3 bispecific antibody on tumor volume in the NCI-H929 human multiple myeloma mouse xenograft tumor model. Invention Details I. Definitions and Explanations Unless otherwise stated, the following terms as used in this disclosure have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0038] The term "antibody" is used in the broadest sense to encompass natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and various antibody structures of single-chain antibodies, as long as they exhibit the desired antigen-binding activity.

[0039] The term "multispecific" refers to an antibody's ability to specifically bind to multiple different antigenic determinants, such as two (i.e., "bispecific") or more different antigenic determinants. In this article, antigenic determinant is synonymous with antigenic epitope. Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different antigenic determinant. Different antigenic determinants can be expressed on the same or different cells. Different antigenic determinants can differ depending on the antigen (e.g., binding to antigens GPRC5D and CD3) or can be present on the same antigen. An antigenic determinant is a specific chemical group with a certain composition and structure on the surface or other sites of an antigen molecule, capable of specifically binding to its corresponding antibody or sensitized lymphocyte. An example of an antigenic determinant is GPRC5D, which has multiple antigenic determinants with known or unknown structures. A specific bispecific antibody, for example, can bind to GPRC5D and CD3.

[0040] The term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. Specific antigen-binding moieties can be, for example, Fab, scFv, or scFab.

[0041] The terms “first,” “second,” or “third” used in this disclosure to refer to antigen-binding portions, antigens, Fc polypeptides, peptide linkers, polypeptide chains, etc., are used for ease of distinction when more than one type of portion is present. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order or orientation.

[0042] The term "fusion" means that components (e.g., antigen-binding moieties, Fc peptides, etc.) are linked directly or via one or more peptide linkers through peptide bonds. For example, some peptide linkers consist of 1 to 50 amino acids linked by peptide bonds, wherein said amino acids may be selected from 20 naturally occurring amino acids; in a more preferred embodiment, the 1 to 50 amino acids are selected from glycine, alanine, proline, serine, asparagine, glutamine, and lysine.

[0043] The term "variable domain" or "variable region" refers to a domain of an antibody involved in the binding of the antibody to an antigen. For example, natural four-chain antibodies (e.g., derived from humans, mice, etc.) have a heavy chain variable domain (also called a heavy chain variable domain, VH, or VH domain) and a light chain variable domain (also called a light chain variable domain, VL, or VL domain). In most cases, each variable domain of a natural antibody essentially consists of four "frame regions (FRs)" and three "complementarity-determining regions (CDRs)." The four frame regions are referred to as frame region 1 (or FR1), frame region 2 (or FR2), frame region 3 (or FR3), and frame region 4 (or FR4), respectively; these frame regions are separated by three complementarity-determining regions, referred to in the art and hereinafter as complementarity-determining regions 1 (or CDR1), 2 (or CDR2), and 3 (or CDR3), respectively. Therefore, the general structure of a variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Variable domains endow antibodies with antigen specificity because they have antigen-binding sites.

[0044] The term "complementarity-determining region" (CDR) is also known as the "hypervariant region" (HVR). Natural four-chain antibodies typically contain six CDRs: three in the heavy chain variable region, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3); and three in the light chain variable region, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3).

[0045] Currently, there are many methods for defining CDRs. The Kabat definition, based on sequence variability, is the most commonly used (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); while the Chothia definition is based on the location of structural loops (Cyrus Chothia, et al, Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901-917 (1987)). The AbM definition is a compromise between the Kabat and Chothia definitions and is used by the AbM antibody modeling software from Oxford Molecular. The "contact" definition of CDRs is based on the analysis of available complex crystal structures. Additionally, there is the CCG definition. However, it should be noted that the boundaries of the CDRs (Continuous Derivatives) of the same antibody variable region obtained by different methods may differ; that is, the CDR sequences of the same antibody variable region defined by different methods may be different. Therefore, when referring to antibodies defined by a specific CDR sequence, the scope of the antibody also includes antibodies defined by CDR sequences of other arbitrary definitions (e.g., one or more combinations of definitions such as Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0046] The term "Fab" refers to a protein composed of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of an immunoglobulin. In this text, Fab refers to the Fab molecule in its native form or modified form, i.e., a Fab heavy chain (VH-CH1, N-to-C-terminal) consisting of the VH variable region and CH1 constant region of the heavy chain, and a Fab light chain (VL-CL, N-to-C-terminal) consisting of the VL variable region and CL constant region of the light chain. A modified Fab can be, for example, a Fab with amino acid substitutions introduced into the CH1 / CL domain and / or the VH / VL domain. A specific example of a modified Fab is a Fab with amino acid substitutions introduced into the CL domain.

[0047] The term "scFv" encompasses the VH and VL domains of an immunoglobulin, where these domains are present within a single polypeptide chain. In some embodiments, the scFv also includes a peptide linker between the VH and VL domains, enabling the scFv to form the structure required for antigen binding. In a specific example, the scFv is arranged from the N-terminus to the C-terminus in the following order: VL-linker-VH.

[0048] The terms “Fc domain,” “Fc,” or “Fc region” are used herein to define the C-terminal region of the immunoglobulin heavy chain, which contains at least a portion of the constant region. This term includes both native sequence Fc and variant Fc. The C-terminal lysine residue (Lys447) of the Fc may or may not be present. Unless otherwise stated, the amino acid residues in the Fc or constant region are numbered according to the EU numbering system, also known as the EU index, described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. As used herein, one “Fc polypeptide” of the Fc domain refers to one of the two polypeptides that form the dimer Fc domain. For example, the Fc polypeptide of the IgG Fc domain contains IgG CH2 and IgG CH3.

[0049] The term “treatment” means administering the compounds (e.g., monospecific antibodies, bispecific antibodies) or pharmaceutical compositions described in this disclosure to prevent, improve, or eliminate a disease or one or more symptoms associated with said disease, including but not limited to: (i) preventing the occurrence of a disease or disease state in mammals, particularly when such mammals are susceptible to the disease state but have not yet been diagnosed with it; (ii) suppressing a disease or disease state, i.e., halting its development; (iii) alleviating a disease or disease state, even if the disease or disease state subsides; and (iv) reducing any direct or indirect pathological consequences of the disease or disease state.

[0050] The term "therapeutic effective amount" means (i) the amount of the disclosed compound used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the monospecific antibody, bispecific antibody, antibody-drug conjugate, engineered immune effector cell, or pharmaceutical composition of the present disclosure constituting a "therapeutic effective amount" may vary depending on factors such as the compound or pharmaceutical composition and its ability to elicit a desired response in an individual, the disease state and its severity, the route of administration, and the age, sex, and weight of the mammal to be treated. Therapeutic effective amounts may also be routinely determined by those skilled in the art based on their own knowledge and the content of this disclosure.

[0051] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0052] The term "excipient" refers to any component other than the active ingredient (e.g., the antibody of this disclosure). The selection of excipients will depend to a great extent on factors such as the specific method of administration, the efficacy of the excipient in terms of solubility and stability, and the nature of the dosage form.

[0053] The term "isolated" refers to a target compound that has been isolated from its natural environment, such as an antibody or its antigen-binding fragment or nucleic acid.

[0054] Term "X" n "Xaa" is equivalent to "Xaa" and refers to unspecified amino acids, the scope of which is specified by subsequent definitions in the relevant description.

[0055] As used in this article, the term "EC" 50 "Colony" refers to the half-maximal effective concentration, which is the antibody concentration at which the induced response is 50% of the maximum response, i.e., half the distance between the maximum response and the baseline. 50 It can be measured by ELISA or FACS analysis or any other method known in the art.

[0056] “K D "Refers to the equilibrium dissociation constant, which is the self-dissociation rate constant (k)" d ) relative to the binding rate constant (k a (i.e., k) d / k aThe ratio of K to α is expressed as molar concentration (M). The K of the antibody D The value can be determined using methods well-established in the art. The K value used for antibody determination... D The preferred method is to use surface plasmon resonance (SPR) technology, preferably using a biosensor system such as the Biacore surface plasmon resonance system for analysis.

[0057] The term "identity," also known as consistency, refers to the percentage of amino acid residues in the sequence to be aligned that are identical to those in the specific amino acid sequence shown herein, after aligning the sequence to be aligned with it and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Amino acid sequence alignment for identity can be performed using various methods within the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the aligned sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0058] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Preferably, the subject according to this disclosure is a human. Unless otherwise stated, the terms "patient" and "subject" may be used interchangeably. "Subject in need" includes subjects who already have a disease or condition, subjects at risk of developing a disease or condition, and subjects who may have a disease or condition and whose purpose is to prevent, delay, or reduce a disease or condition.

[0059] As used herein, “about” means within the acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may, in accordance with art practice, mean within one or more standard deviations. Alternatively, “about” may mean a range of up to ±5%, such as fluctuations within ±2%, ±1%, or ±0.5% of a given specific numerical range. When a particular value is given in the scope of this disclosure, unless otherwise stated, “about” shall be understood to mean within the acceptable range of error for that particular value. In this document, unless otherwise stated, the values ​​of step parameters or conditions are implicitly modified by “about”.

[0060] The terms “comprise,” “containing,” and “comprising” and their equivalents (e.g., contain, contains, containing, include, includes, and including) shall be understood as “including but not limited to,” meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be covered.

[0061] In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa.

[0062] II. Monospecific antibodies or their antigen-binding moieties This disclosure provides a monospecific antibody or antigen-binding portion thereof that binds to GPRC5D, comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, 10, 18, 26 or 34; HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, 11, 19, 27 or 35; LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, 13, 21, 29 or 37; and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 30 or 38.

[0063] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; wherein X1 is selected from D or E, and X2 is selected from G or A. In some specific embodiments, X1 is D and X2 is G. In some specific embodiments, X1 is D and X2 is A. In some specific embodiments, X1 is E and X2 is G.

[0064] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0065] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 60, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0066] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 61, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0067] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0068] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22.

[0069] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30.

[0070] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0071] This disclosure also provides a monospecific antibody or antigen-binding portion thereof that binds to GPRC5D, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, 15, 23, 31, 39, 66 or 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8, 16, 24, 32, 40, 67 or 69.

[0072] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8.

[0073] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 16.

[0074] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24.

[0075] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 32.

[0076] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 39, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 40.

[0077] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0078] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 63, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0079] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0080] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 65, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0081] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding moiety comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; wherein X3 is selected from Q or V, X4 is selected from P or A, X5 is selected from L or V, X6 is selected from K or A, X7 is selected from I or M, X8 is selected from D or E, X9 is selected from G or A, X... 10 Selected from K or R, X 11 Selected from A or V, X 12 Selected from K or T, X 13 Selected from S or A, X 14 Selected from T or R, X 15 Selected from S or T, X 16 Choose from A or Q.

[0082] In some embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 69.

[0083] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 7, 15, 23, 31, 39, 62, 63, 64, 65, 66, or 68, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 8, 16, 24, 32, 40, 67, or 69.

[0084] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0085] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 16. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0086] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 24. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22.

[0087] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 32. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30.

[0088] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 40. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0089] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0090] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0091] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 60, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0092] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 61, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0093] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67; wherein X3 is selected from Q or V, X4 is selected from P or A, X5 is selected from L or V, X6 is selected from K or A, X7 is selected from I or M, X8 is selected from D or E, X9 is selected from G or A, X 10 Selected from K or R, X 11 Selected from A or V, X 12 Selected from K or T, X 13 Selected from S or A, X 14 Selected from T or R, X 15 Selected from S or T, X 16 The amino acid sequence is selected from A or Q. Further, in some such embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; wherein X1 is selected from D or E, and X2 is selected from G or A.

[0094] In some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 69. Furthermore, in some such embodiments, the monospecific antibody binding GPRC5D or its antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0095] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0096] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16.

[0097] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24.

[0098] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 32.

[0099] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40.

[0100] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0101] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0102] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0103] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0104] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0105] In some embodiments, the monospecific antibody that binds to GPRC5D or its antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69.

[0106] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 7 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 8.

[0107] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 15 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 16.

[0108] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 23 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 24.

[0109] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 31 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 32.

[0110] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 39 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 40.

[0111] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 62 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0112] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 63 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0113] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 64 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0114] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 65 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0115] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 66 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0116] In some specific embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 68 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 69.

[0117] In this disclosure, the monospecific antibody binding to GPRC5D or its antigen-binding portion thereof is murine, chimeric, or humanized. Humanization can reduce immunogenicity. Therefore, in some embodiments, the monospecific antibody binding to GPRC5D or its antigen-binding portion thereof is humanized.

[0118] In some embodiments, the monospecific antibody binding to GPRC5D of this disclosure may further comprise a constant region of an immunoglobulin, or a fragment, analogue, variant, or derivative of said constant region. In some embodiments, the constant region comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is derived from the heavy chain of human immunoglobulins, such as IgG1, IgG2, IgG3, and IgG4, or the heavy chain of other classes of immunoglobulins. In some embodiments, the light chain constant region is derived from the light chain of human immunoglobulins, such as the κ or λ light chain of human immunoglobulins. In some embodiments, the constant region may contain any modification of this disclosure or known in the art, such as the insertion, deletion, substitution, or chemical modification of amino acids. In some embodiments, the constant region contains mutations that alter effector function. In some embodiments, any amino acid residue of the constant region may be substituted with any allotype amino acid residue. In some embodiments, the C-terminal lysine (Lys447) of the heavy chain constant region may or may not be present. In some specific embodiments, the GPRC5D-binding monospecific antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 70, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the GPRC5D-binding monospecific antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 72, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the GPRC5D-binding monospecific antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 74, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the GPRC5D-binding monospecific antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 76, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the GPRC5D-binding monospecific antibody comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 80, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 82. In some implementations, the C-terminal lysine residue of the heavy chain is absent.

[0119] Tables S1 and S2 provide amino acid sequence information for the CDR and variable regions of several exemplary monospecific antibodies that bind to GPRC5D (e.g., chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, and Chi-169, and humanized antibodies hu131-v1, hu131-v2, hu131-v3, hu131-v4, and hu128-v1). These exemplary antibodies can be used to construct the bispecific antibodies of this disclosure.

[0120] Table S1. Amino acid sequences of CDR and variable regions (SEQ ID NO:)

[0121] The monospecific antibody or its antigen-binding moiety that binds to GPRC5D disclosed herein can bind to human GPRC5D and / or monkey GPRC5D and / or mouse GPRC5D. In some embodiments, the monospecific antibody or its antigen-binding moiety does not bind or substantially does not bind to GPRC5A, GPRC5B, and GPRC5C.

[0122] isolated nucleic acids This disclosure provides isolated nucleic acids comprising nucleotide sequences encoding a monospecific antibody or antigen-binding moiety thereof that binds to GPRC5D as described in this disclosure. The sequence listing provides exemplary examples of nucleotide sequences encoding monospecific antibodies or antigen-binding moieties thereof that bind to GPRC5D.

[0123] carrier This disclosure provides a vector comprising the described nucleic acid. In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector, a specific example being pcDNA3.1(+). The expression vector may optionally be any expression vector capable of expressing the monospecific antibody binding GPRC5D described in this disclosure.

[0124] host cells This disclosure provides host cells comprising the nucleic acids or vectors of this disclosure. In some embodiments, the host cell is a suitable host cell for cloning or expressing a monospecific antibody or its antigen-binding portion. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing monospecific antibodies or their antigen-binding portions. Mammalian cells include, for example, Chinese hamster ovary (CHO) cells and CHO-S cells.

[0125] Antibody-drug conjugates (ADCs) This disclosure provides antibody-drug conjugates comprising a monospecific antibody or antigen-binding moiety thereof that binds to GPRC5D. In some embodiments, the antibody-drug conjugate comprises the monospecific antibody or antigen-binding moiety thereof that binds to GPRC5D, and a cytotoxic drug. The monospecific antibody or antigen-binding moiety thereof that binds to GPRC5D and the cytotoxic drug are preferably linked by a linker, which may be a cleavable linker or a non-cleavable linker.

[0126] Chimeric antigen receptor (CAR) This disclosure also provides an anti-GPRC5D chimeric antigen receptor (CAR) comprising a monospecific antibody or antigen-binding portion thereof that binds to GPRC5D.

[0127] Anti-GPRC5D CARs may include (a) an extracellular antigen-binding domain containing GPRC5D binding; (b) a transmembrane domain; and (c) an intracellular signal transduction domain.

[0128] This disclosure also provides engineered immune effector cells that contain the CAR of this disclosure. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells.

[0129] Pharmaceutical Composition This disclosure provides pharmaceutical compositions comprising a monospecific antibody or antigen-binding moiety thereof that binds to GPRC5D, an antibody-drug conjugate, or engineered immune effector cells, and further comprising one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0130] Methods for preparing monospecific antibodies or their antigen-binding moieties In some embodiments, this disclosure provides a method for preparing a monospecific antibody or antigen-binding moiety thereof that binds to GPRC5D, the method comprising: culturing the host cells to express the monospecific antibody or antigen-binding moiety thereof, and isolating and purifying the monospecific antibody or antigen-binding moiety thereof in the system. To generate the monospecific antibody or antigen-binding moiety thereof, nucleic acid encoding the monospecific antibody or antigen-binding moiety thereof is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The nucleic acid can be obtained using various methods well known in the art, such as gene splicing and chemical synthesis.

[0131] The purity of a monospecific antibody or its antigen-binding moiety can be determined using any of a variety of well-known analytical methods, such as gel electrophoresis, high-performance liquid chromatography, size exclusion chromatography, etc.

[0132] The physicochemical properties and / or biological activities of the monospecific antibodies or their antigen-binding moieties disclosed herein can be identified, screened, or characterized by a variety of assay methods known in the art.

[0133] use This disclosure provides the use of a monospecific antibody or its antigen-binding moiety that binds to GPRC5D. In some specific embodiments, the monospecific antibody used may be Chi-89, Chi-105, Chi-128, Chi-131, Chi-169, hu131-v1, hu131-v2, hu131-v3, hu131-v4, or / and hu128-v1.

[0134] This disclosure provides the use of the monospecific antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immune effector cells, or the pharmaceutical composition in the preparation of a medicament for treating diseases expressing GPRC5D. In some embodiments, the monospecific antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immune effector cells, or the pharmaceutical composition are prepared with one or more additional therapeutic agents. The additional therapeutic agents may be known in the art as oncology therapeutic agents or autoimmune disease therapeutic agents.

[0135] This disclosure provides a method for treating a disease expressing GPRC5D, comprising administering to a subject in need a therapeutically effective amount of the monospecific antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immune effector cells, or the pharmaceutical composition. In some embodiments, the disease is a tumor or an autoimmune disease.

[0136] In some embodiments, the tumor is a non-solid tumor. In some embodiments, the tumor is a hematologic malignancy. In some embodiments, the tumor is a B-cell lymphoma. In some embodiments, the tumor is a GPRC5D-expressing B-cell lymphoma. In some embodiments, the tumor is multiple myeloma (MM). In some embodiments, the tumor is a GPRC5D-expressing multiple myeloma.

[0137] In some implementations, the autoimmune disease is, for example, systemic lupus erythematosus and / or rheumatoid arthritis.

[0138] III. Bispecific antibodies This disclosure provides a bispecific antibody comprising a first antigen-binding portion that binds to GPRC5D and a second antigen-binding portion that binds to CD3, wherein the first antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 59, 10, 18, 26 or 34; HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, 11, 19, 27 or 35; LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, 13, 21, 29 or 37; and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 30 or 38.

[0139] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; wherein X1 is selected from D or E, and X2 is selected from G or A. In some specific embodiments, X1 is D and X2 is G. In some specific embodiments, X1 is D and X2 is A. In some specific embodiments, X1 is E and X2 is G.

[0140] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0141] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 60, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0142] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 61, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0143] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0144] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22.

[0145] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30.

[0146] In some embodiments, the first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

[0147] This disclosure also provides a bispecific antibody comprising a first antigen-binding portion that binds to GPRC5D and a second antigen-binding portion that binds to CD3, wherein the first antigen-binding portion comprises: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, 15, 23, 31, 39, 66 or 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8, 16, 24, 32, 40, 67 or 69.

[0148] In some embodiments, the first antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8.

[0149] In some embodiments, the first antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 16.

[0150] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24.

[0151] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 32.

[0152] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 39, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 40.

[0153] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0154] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 63, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0155] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0156] In some embodiments, the first antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 65, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.

[0157] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; wherein X3 is selected from Q or V, X4 is selected from P or A, X5 is selected from L or V, X6 is selected from K or A, X7 is selected from I or M, X8 is selected from D or E, X9 is selected from G or A, X... 10 Selected from K or R, X 11 Selected from A or V, X 12 Selected from K or T, X 13 Selected from S or A, X 14 Selected from T or R, X 15 Selected from S or T, X 16 Choose from A or Q.

[0158] In some embodiments, the first antigen-binding region comprises: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 69.

[0159] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 7, 15, 23, 31, 39, 62, 63, 64, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8, 16, 24, 32, 40, 67, or 69, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8, 16, 24, 32, 40, 67, or 69.

[0160] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0161] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 16. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 14.

[0162] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 24. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 18, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 19, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 22.

[0163] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 32. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 26, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 28, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30.

[0164] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 40. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38.

[0165] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0166] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0167] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 60, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0168] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 61, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0169] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67; wherein X3 is selected from Q or V, X4 is selected from P or A, X5 is selected from L or V, X6 is selected from K or A, X7 is selected from I or M, X8 is selected from D or E, X9 is selected from G or A, X 10 Selected from K or R, X 11 Selected from A or V, X 12 Selected from K or T, X 13 Selected from S or A, X 14 Selected from T or R, X 15 Selected from S or T, X 16The amino acid sequence is selected from A or Q. Further, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; wherein X1 is selected from D or E, and X2 is selected from G or A.

[0170] In some embodiments, the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 69. Furthermore, in some such embodiments, the first antigen-binding portion further comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38.

[0171] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0172] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16.

[0173] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24.

[0174] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 32.

[0175] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40.

[0176] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0177] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0178] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0179] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0180] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0181] In some embodiments, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69.

[0182] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 7 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 8.

[0183] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 15 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 16.

[0184] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 23 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 24.

[0185] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 31 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 32.

[0186] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 39 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 40.

[0187] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 62 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0188] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 63 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0189] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 64 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0190] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 65 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0191] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 66 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 67.

[0192] In some specific embodiments, the first antigen-binding portion includes the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 68 and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 69.

[0193] In this disclosure, the first antigen-binding portion is murine, chimeric, or humanized. Humanization can reduce immunogenicity. Therefore, in some embodiments, the first antigen-binding portion is humanized.

[0194] The exemplary monospecific antibodies provided in Tables S1 and S2 can be used to construct the bispecific antibodies of this disclosure. For example, in some embodiments, the CDR of the first antigen-binding portion of the bispecific antibody is selected from the six CDRs of any exemplary GPRC5D-binding monospecific antibody. For example, in some embodiments, the variable region of the first antigen-binding portion of the bispecific antibody is selected from the heavy chain variable region and the light chain variable region of any exemplary GPRC5D-binding monospecific antibody.

[0195] In the bispecific antibody disclosed herein, the second antigen-binding moiety provides the ability to target CD3. The second antigen-binding moiety can be Fab, scFv, or scFab (single-chain Fab). In some embodiments, the second antigen-binding moiety is a CD3-binding scFv.

[0196] In some embodiments, the second antigen-binding portion is murine, chimeric, or humanized. In some embodiments, the second antigen-binding portion is humanized. In some specific embodiments, both the first and second antigen-binding portions are humanized.

[0197] In some embodiments, the second antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 94, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 95, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 96, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 97, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 98, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 99.

[0198] In some embodiments, the second antigen-binding portion comprises: HCDR1, HCDR2, and HCDR3 in the variable region of the heavy chain as shown in SEQ ID NO: 100, and LCDR1, LCDR2, and LCDR3 in the variable region of the light chain as shown in SEQ ID NO: 101.

[0199] In some embodiments, the second antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 100, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 101. In some embodiments, the second antigen-binding portion comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 100, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 101. In some embodiments, the second antigen-binding portion comprises a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 100 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 101.

[0200] The bispecific antibody disclosed herein can bind to GPRC5D. In some embodiments, the bispecific antibody can bind to human GPRC5D and / or monkey GPRC5D and / or mouse GPRC5D. In some embodiments, the bispecific antibody does not bind or substantially does not bind to GPRC5A, GPRC5B, and GPRC5C.

[0201] In some specific examples, the bispecific antibody disclosed herein may be GC35 or GC39.

[0202] The bispecific antibody disclosed herein can target GPRC5D expressed on the surface of tumor cells and can also bind to T cells, inducing T cell activation and activating the tumor-killing activity of T cells. The bispecific antibody of this disclosure exhibits excellent anti-tumor properties, such as lysing tumor cells or inhibiting tumor cell proliferation. In some embodiments, the bispecific antibody has excellent anti-tumor properties against tumors such as multiple myeloma.

[0203] The molecular design of the bispecific antibody disclosed herein is easy to express and purify.

[0204] The bispecific antibody disclosed herein has good safety.

[0205] Bispecific antibody configuration The bispecific antibody disclosed herein may be Fc domain-free, and the first antigen-binding portion and the second antigen-binding portion are fused through a suitable linker.

[0206] The bispecific antibody disclosed herein may have an Fc domain, which can extend the half-life and provide Fc domain-related effectors, among other functions. In some embodiments, the first antigen-binding portion and / or the second antigen-binding portion of the bispecific antibody disclosed herein may be fused to the N-terminus of the Fc domain.

[0207] In the bispecific antibody disclosed herein, the first antigen-binding portion and the second antigen-binding portion can adopt any suitable structural form. In some embodiments, the first antigen-binding portion and the second antigen-binding portion can each independently be Fab, scFv, scFab, or other structures.

[0208] The bispecific antibodies disclosed herein can be multivalent, such as bivalent, tetravalent, etc. In some embodiments, the bispecific antibody is bivalent, meaning that both the first antigen-binding portion and the second antigen-binding portion each provide monovalent binding to the corresponding antigen.

[0209] In some implementations, the first antigen-binding portion is Fab, and the second antigen-binding portion is scFv.

[0210] In some embodiments, the bispecific antibody disclosed herein further comprises an Fc domain consisting of two Fc polypeptides.

[0211] In some embodiments, the first antigen-binding moiety is Fab, the second antigen-binding moiety is scFv, the first antigen-binding moiety is fused at the C-terminus of its Fab heavy chain to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding moiety is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain. Such a configuration is schematically described in... Figure 9 In this embodiment, and more specifically, the bispecific antibody consists of three polypeptide chains, wherein the first polypeptide chain contains the Fab light chain of the first antigen-binding moiety, the second polypeptide chain contains the Fab heavy chain of the first antigen-binding moiety and one of the Fc polypeptides of the Fc domain, and the third polypeptide chain contains the second antigen-binding moiety and another Fc polypeptide of the Fc domain.

[0212] In the bispecific antibody disclosed herein, the heavy chain variable region and the light chain variable region of the second antigen-binding moiety are linked by a peptide linker to form an scFv. The peptide linker can be any suitable linker, such as charged and / or flexible.

[0213] In some embodiments, the second antigen-binding moiety is scFv, with a VH-VL structure from the N-terminus to the C-terminus. In some embodiments, the second antigen-binding moiety is scFv, with a VL-VH structure from the N-terminus to the C-terminus. Optionally, the heavy chain variable region of the second antigen-binding moiety is fused to the light chain variable region of the second antigen-binding moiety via a peptide linker.

[0214] In some specific embodiments, the peptide linker consists of 1 to 50 amino acids linked by peptide bonds, wherein said amino acids may be selected from 20 naturally occurring amino acids. In some more preferred embodiments, the 1 to 50 amino acids are selected from glycine, alanine, proline, serine, asparagine, glutamine, and lysine. Thus, exemplary peptide linkers may be polyglycine (especially (Gly)4, (Gly)5), poly(Gly-Ser), (Gly)3AsnGlySer(Gly)2, (Gly)3Cys(Gly)4, GlyProAsnGlyGly, or those disclosed in Table 4 of patent application WO2019195535, etc.

[0215] In some embodiments, the peptide linker may be a peptide linker composed of glycine and serine. In some embodiments, the peptide linker may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acids. In some embodiments, the peptide linker comprises peptide linkers in units of GGGGS. In some embodiments, the peptide linker in units of GGGGS is (GGGGS). n Where n is any number between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or any range defined by any two of the aforementioned numbers, such as 1-5, 2-5, 3-6, 2-4, 1-4, etc. In some specific embodiments, the peptide linker is a linker polypeptide comprising GGGGS (SEQ ID NO: 104), (GGGGS)2, (GGGGS)3, or (GGGGS)4. In some specific embodiments, the heavy chain variable region of the second antigen-binding moiety is fused to the light chain variable region of the second antigen-binding moiety via the peptide linker (GGGGS)3.

[0216] In some specific implementations, the first antigen-binding region is Fab, the second antigen-binding region is scFv, the first antigen-binding region is fused at the C-terminus of its Fab heavy chain to the N-terminus of one of the Fc peptides in the Fc domain, the heavy chain variable region of the second antigen-binding region is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain, and the light chain variable region of the second antigen-binding region is fused at its C-terminus to the heavy chain variable region of the second antigen-binding region; preferably, the heavy chain variable region of the second antigen-binding region is fused to the light chain variable region of the second antigen-binding region via a peptide linker (GGGGS)3.

[0217] When fused with Fc, fusion is typically via the hinge region. In one embodiment, the first antigen-binding moiety is fused to one of the Fc peptides in the Fc domain via a first hinge, and the second antigen-binding moiety is fused to another Fc peptide in the Fc domain via a second hinge. In some embodiments, the first and second hinges are capable of forming covalent bonds, such as disulfide bonds, with each other. The first and / or second hinges may contain amino acids from the hinge region of human IgG, which contains a native hinge region or a variant thereof. In some embodiments, the first and / or second hinges contain amino acids from the hinge region of human IgG1. In some embodiments, the first and / or second hinges contain amino acids from the hinge region of human IgG4. In some specific embodiments, the first hinge contains EPKSCDKTHTCPPCP (SEQ ID NO: 102), and the second hinge contains GEPKSSDKTHTCPPCP (SEQ ID NO: 103).

[0218] In other embodiments, the first antigen-binding moiety is Fab, the second antigen-binding moiety is scFv, the first antigen-binding moiety is fused at the C-terminus of its Fab light chain to the N-terminus of one of the Fc peptides in the Fc domain, and the second antigen-binding moiety is fused at its C-terminus to the N-terminus of another Fc peptide in the Fc domain.

[0219] Fc domain The Fc domain of a bispecific antibody consists of a pair of polypeptide chains containing heavy chain domains of immunoglobulin molecules. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each Fc polypeptide containing CH2 and CH3 of the constant region of the IgG heavy chain. The two Fc polypeptides of the Fc domain can stably associate with each other. In some embodiments, the bispecific antibody of this disclosure comprises a single Fc domain.

[0220] In some embodiments, the Fc domain of the bispecific antibody is an IgG Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain. In some specific embodiments, the Fc domain is a human IgG1 Fc domain.

[0221] In some embodiments, the Fc domain contains modifications, such as amino acid substitutions. These modifications may include, for example, modifications that promote heterodimerization, alter effector function, or change the binding affinity to protein A.

[0222] In some implementations, the Fc includes modifications that promote heterodimerization.

[0223] The bispecific antibody disclosed herein comprises a different antigen-binding moiety fused to one or the other of two Fc polypeptides in the Fc domain; therefore, the two Fc polypeptides are typically contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides yield several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibody in recombinant production, it is advantageous to introduce modifications into the Fc domain of the bispecific antibody that promote the binding of the desired polypeptide. Therefore, in a specific embodiment, the Fc domain comprises amino acid substitutions that promote association between the two Fc polypeptides in the Fc domain.

[0224] The most extensive protein-protein interaction between the two Fc polypeptides of the human IgG Fc domain occurs in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is performed in the CH3 domain of the Fc domain.

[0225] In a specific implementation, this modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two Fc polypeptides in the Fc domain and a "mortar" modification in the other of the two Fc polypeptides in the Fc domain. Typically, this method involves introducing a protrusion ("knob") at the interface of one Fc polypeptide and a corresponding depression ("mortar") at the interface of the other Fc polypeptide, such that the protrusion can be positioned within the depression to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of one Fc polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary depression of the same or similar size as the protrusion is created at the interface of the other Fc polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0226] Therefore, in a specific implementation, in the CH3 domain of one Fc peptide of the bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the Fc peptide that can be positioned in the CH3 domain of the other Fc peptide. In the CH3 domain of the other Fc peptide, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a depression in the CH3 domain of the Fc peptide.

[0227] In some embodiments, according to EU designations, one Fc polypeptide of the Fc domain comprises amino acid substitutions of 354C and / or 366Y / W, and the other Fc polypeptide comprises amino acid substitutions of 349C, 366S, 368A, and / or 407T / V. In some specific embodiments, according to EU designations, one Fc polypeptide of the Fc domain comprises amino acid substitutions of 354C and 366Y / W, and the other Fc polypeptide comprises amino acid substitutions of 349C, 366S, 368A, and 407T / V. In some more specific embodiments, according to EU designations, one Fc polypeptide of the Fc domain comprises amino acid substitutions of 354C and 366W, and the other Fc polypeptide comprises amino acid substitutions of 349C, 366S, 368A, and 407V. In the above embodiments, the Fc may be human IgG1 Fc. In one specific implementation, according to the EU designation, one of the Fc polypeptides in the Fc domain comprises amino acid substitutions S354C and T366W, and the other Fc polypeptide comprises amino acid substitutions Y349C, T366S, L368A, and Y407V.

[0228] In some embodiments, the Fc domain includes modifications that alter effector function. In some specific embodiments, the Fc domain of the bispecific antibody is modified to reduce the binding affinity of the Fc domain to the Fc receptor and / or decrease effector function compared to the unmodified Fc domain. Reducing the binding affinity of the Fc domain to the Fc receptor and / or decreasing effector function is beneficial for improving cytokine release and side effects.

[0229] In some embodiments, modifications that reduce the binding affinity of the Fc domain to the Fc receptor and / or decrease effector function are amino acid substitutions. In some embodiments, the Fc domain contains amino acid substitutions at one or more positions selected from 233, 234, 235, 297, 331, and 329. In some embodiments, the Fc domain contains amino acid substitutions at one or more positions selected from 234, 235, and 329. In some embodiments, the Fc domain contains amino acid substitutions 234A and 235A. In one such embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc. In some embodiments, the Fc domain contains an amino acid substitution at position 329. In a more specific embodiment, the amino acid substitution is 329A, 329R, or 329G. In some embodiments, the Fc domain contains an amino acid substitution at position 329 and additional amino acid substitutions selected from positions 233, 234, 235, 297, and 331. In a more specific embodiment, the additional amino acid substitution is 233P, 234A, 235A, 235E, 297A, 297D, or / and 331S. In some embodiments, the Fc domain contains amino acid substitutions at positions 329, 234, and 235. In a more specific embodiment, the Fc domain contains amino acid substitutions 234A, 235A, and 329G. In another more specific embodiment, the Fc domain contains amino acid substitutions 234A, 235A, and 329A. In one such embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc. In one specific embodiment, the Fc domain contains amino acid substitutions L234A, L235A, and P329G. In another specific embodiment, the Fc domain contains amino acid substitutions L234A, L235A, and P329A.

[0230] In some specific embodiments, the Fc domain is a human IgG1 Fc domain containing amino acid substitutions for L234A, L235A, and P329A / G / R. Further, the Fc domain is a human IgG1 Fc domain containing amino acid substitutions for L234A, L235A, and P329A.

[0231] The amino acid substitutions described above that reduce the binding affinity of the Fc domain to the Fc receptor and / or decrease effector function occur on both polypeptide chains of the Fc domain.

[0232] In some embodiments, the Fc domain comprises a modification that reduces or eliminates the binding of the CH3 region of one Fc polypeptide to protein A (from Staphylococcus aureus). In some embodiments, the Fc domain comprises an amino acid substitution that reduces or eliminates the binding of the CH3 region of one Fc polypeptide to protein A. In some embodiments, according to EU designations, the Fc domain comprises an amino acid substitution (a) 435R or (b) 435R and 436F, which occurs only on one Fc polypeptide and not on the other. In some embodiments, according to EU designations, the Fc domain comprises an amino acid substitution (a) 435R or (b) 435R and 436F occurring only on one Fc polypeptide. In some specific embodiments, according to EU designations, the Fc domain comprises an amino acid substitution H435R and Y436F occurring only on one Fc polypeptide. In some specific embodiments, according to EU designations, the Fc domain comprises an amino acid substitution H435R occurring only on one Fc polypeptide. In the above implementation scheme, the Fc is IgG1 Fc, especially human IgG1 Fc.

[0233] In the bispecific antibody disclosed herein, the Fc domain may include one, two, or three of the following modifications: modifications that promote heterodimerization, modifications that alter effector function, and modifications that reduce or eliminate the binding of the CH3 region of an Fc polypeptide within the Fc domain to protein A. In some embodiments, the Fc domain includes modifications that promote heterodimerization, modifications that alter effector function, and modifications that reduce or eliminate the binding of the CH3 region of an Fc polypeptide within the Fc domain to protein A. Combinations of the above different modification types are possible. For example, in one specific embodiment, according to EU designation, the Fc domain includes the following amino acid substitutions: i. 349C, 366S, 368A, 407T / V, 354C and 366Y / W; wherein, amino acid substitutions of 354C and 366Y / W are on the same Fc polypeptide, and are not on the same Fc polypeptide as the other amino acid substitutions in (i); ii.234A, 235A, and 329A; and iii. (a) 435R or (b) 435R and 436F, occurring only on one of the Fc peptides.

[0234] In a more specific embodiment, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide comprises amino acid substitutions: L234A, L235A, P329A, S354C, T366W, and H435R. In this specific embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc.

[0235] In another, more specific embodiment, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide comprises amino acid substitutions: L234A, L235A, P329A, S354C, T366W, H435R, and Y436F. In this particular embodiment, the Fc is IgG1 Fc, particularly human IgG1 Fc.

[0236] In the context of this disclosure, amino acid substitution is represented as: original amino acid - position - substituted amino acid, using a three-letter code (Xaa) or a single-letter code (X) to represent the amino acid residue, and the original amino acid may be omitted. Therefore, for example, "H435R" or "435R" means that the amino acid H at position 435 or the original amino acid is substituted with amino acid R; the substituted amino acid may include more than one, for example, "T366Y / W" means that the amino acid T at position 366 is substituted with amino acid Y or W.

[0237] In some embodiments, the bispecific antibody comprises three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 84; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 78; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 88.

[0238] In some embodiments, the bispecific antibody comprises three polypeptide chains, wherein one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 86; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 82; and a third polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 88.

[0239] In some embodiments, the bispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 84, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 78, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 88.

[0240] In some embodiments, the bispecific antibody comprises three polypeptide chains, one of which contains the amino acid sequence shown in SEQ ID NO: 86, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 82, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 88.

[0241] This disclosure provides exemplary bivalent bispecific antibodies.

[0242] In one example, the bispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in SEQ ID NO: 84, 78, and 88, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are shown in SEQ ID NO: 85, 79, and 89, respectively.

[0243] In one example, the bispecific antibody consists of three polypeptide chains, the amino acid sequences of which are shown in SEQ ID NO: 86, 82, and 88, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are shown in SEQ ID NO: 87, 83, and 89, respectively.

[0244] isolated nucleic acids This disclosure provides isolated nucleic acids containing nucleotide sequences encoding the bispecific antibodies described herein. The sequence listing provides exemplary examples of nucleotide sequences encoding bispecific antibodies.

[0245] carrier This disclosure provides a vector comprising the described nucleic acid. In some embodiments, the vector is a cloning vector; in other embodiments, the vector is an expression vector, and as a specific example, the expression vector is pcDNA3.1(+). The expression vector may optionally be any expression vector capable of expressing the bispecific antibody described in this disclosure.

[0246] host cells This disclosure provides host cells comprising the nucleic acids or vectors of this disclosure. In some embodiments, the host cell is a suitable host cell for cloning or expressing bispecific antibodies. In some embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing bispecific antibodies. Mammalian cells include, for example, Chinese hamster ovary (CHO) cells and CHO-S cells.

[0247] Pharmaceutical Composition This disclosure provides pharmaceutical compositions comprising the bispecific antibody of this disclosure and further comprising one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other reagents.

[0248] Methods for preparing bispecific antibodies In some embodiments, this disclosure provides a method for preparing a bispecific antibody, the method comprising: culturing the host cells to express the bispecific antibody, and isolating and purifying the bispecific antibody in the system. To generate the bispecific antibody, a nucleic acid encoding the bispecific antibody is isolated and inserted into one or more vectors for further cloning and / or expression in the host cells. The nucleic acid can be obtained using various methods well known in the art, such as gene splicing and chemical synthesis.

[0249] The prepared bispecific antibodies can be purified using techniques known in the art, such as high-performance liquid chromatography, ion-exchange chromatography, gel chromatography, affinity chromatography, size exclusion chromatography, ceramic hydroxyapatite (CHT) chromatography, etc. The specific conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and these are readily apparent to those skilled in the art. For affinity chromatography, purification can be performed using an antibody, ligand, receptor, or antigen that binds to the bispecific antibody. For example, for affinity chromatography of the bispecific antibodies of this disclosure, purification can be performed using a matrix containing protein A or protein G. The bispecific antibodies of this disclosure can be purified sequentially using affinity chromatography, ion-exchange chromatography, gel chromatography, and / or CHT chromatography.

[0250] The purity of bispecific antibodies can be determined using any of a variety of well-known analytical methods, such as gel electrophoresis, high-performance liquid chromatography, size exclusion chromatography, etc.

[0251] The physicochemical properties and / or biological activities of the bispecific antibodies disclosed herein can be identified, screened, or characterized by a variety of assay methods known in the art.

[0252] use This disclosure provides for the use of the bispecific antibodies disclosed herein. In some specific embodiments, the bispecific antibody used may be GC35 and / or GC39.

[0253] This disclosure provides the use of the bispecific antibody or the pharmaceutical composition in the preparation of a medicament for treating diseases expressing GPRC5D. In some embodiments, the bispecific antibody or the pharmaceutical composition is prepared with one or more additional therapeutic agents. These additional therapeutic agents may be known in the art as oncology therapeutic agents or autoimmune disease therapeutic agents.

[0254] This disclosure provides a method for treating a disease expressing GPRC5D, comprising administering the bispecific antibody or the pharmaceutical composition to a subject in need. In some embodiments, the disease is a tumor or an autoimmune disease.

[0255] In some embodiments, the tumor is a non-solid tumor. In some embodiments, the tumor is a hematologic malignancy. In some embodiments, the tumor is a B-cell lymphoma. In some embodiments, the tumor is a GPRC5D-expressing B-cell lymphoma. In some embodiments, the tumor is multiple myeloma (MM). In some embodiments, the tumor is a GPRC5D-expressing multiple myeloma.

[0256] In some implementations, the autoimmune disease is, for example, systemic lupus erythematosus and / or rheumatoid arthritis. Detailed Implementation

[0257] This disclosure also provides the following specific implementation schemes, but the scope of protection of this disclosure is not limited thereto: Implementation Scheme 1. A bispecific antibody comprising a first antigen-binding portion binding to GPRC5D and a second antigen-binding portion binding to CD3, wherein the first antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, 59, 10, 18, 26 or 34; HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, 11, 19, 27 or 35; LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, 13, 21, 29 or 37; and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 30 or 38.

[0258] Implementation Scheme 2. The bispecific antibody according to Implementation Scheme 1, wherein the first antigen-binding portion comprises: (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 59, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; wherein X1 is selected from D or E, and X2 is selected from G or A; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, comprising the amino acid sequence shown in SEQ ID NO: 59, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 59, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (3) HCDR2 containing the amino acid sequence shown in SEQ ID NO: 60, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 61, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (5) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 61. LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14;(6) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22; (7) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30; or (8) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 17. HCDR3, containing the amino acid sequence shown in SEQ ID NO: 36, LCDR1, containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3, containing the amino acid sequence shown in SEQ ID NO: 38.

[0259] Implementation Scheme 3. A bispecific antibody comprising a first antigen-binding portion binding to GPRC5D and a second antigen-binding portion binding to CD3, wherein the first antigen-binding portion comprises: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, 15, 23, 31, 39, 66 or 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8, 16, 24, 32, 40, 67 or 69.

[0260] Implementation Scheme 4. The bispecific antibody according to Implementation Scheme 3, wherein the first antigen-binding portion comprises: (1) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8; (2) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 16; (3) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24; (4) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 31. (5) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 39, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 40; (6) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 62, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (7) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 63, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (8) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 64, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 64. (9) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 65, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.(10) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 66, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67, wherein X3 is selected from Q or V, X4 is selected from P or A, X5 is selected from L or V, X6 is selected from K or A, X7 is selected from I or M, X8 is selected from D or E, X9 is selected from G or A, X; 10 Selected from K or R, X 11 Selected from A or V, X 12 Selected from K or T, X 13 Selected from S or A, X 14 Selected from T or R, X 15 Selected from S or T, X 16 Selected from A or Q; or (11) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 69.

[0261] Implementation Scheme 5. A bispecific antibody according to any one of Implementation Schemes 1-4, wherein the first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 7, 15, 23, 31, 39, 62, 63, 64, 65, 66, or 68, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8, 16, 24, 32, 40, 67, or 69 ....

[0262] Implementation Scheme 6. A bispecific antibody according to any one of Implementation Schemes 1-5, wherein the first antigen-binding portion comprises: (1) a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8; (2) a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in NO:15 has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:16; (3) the amino acid sequence has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:23; and the amino acid sequence has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:23. The amino acid sequence shown in SEQ ID NO: 31 has a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; (4) a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 32; and a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 32.(5) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 40; (6) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 62, and a light ... The amino acid sequence shown in SEQ ID NO: 67 has a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 63; (7) a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; and a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; (8) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; The amino acid sequence shown in SEQ ID NO: 64 has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67;(9) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (10) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 66, and a light ...7. The amino acid sequence shown in SEQ ID NO: 67 has a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 68; or (11) a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 69; and a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 69.

[0263] Implementation Scheme 7. A bispecific antibody according to any one of Implementation Schemes 1-6, wherein the first antigen-binding portion comprises: (1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; (2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16; (3) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24; (4) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 32; (5) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40; (6) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62. (7) A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 63 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (8) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (9) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 65 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (10) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; or (11) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69.

[0264] Implementation Scheme 8. The bispecific antibody according to Implementation Scheme 1, wherein the first antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0265] Implementation Scheme 9. The bispecific antibody according to Implementation Scheme 8, wherein the first antigen-binding portion comprises: a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; preferably, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67.

[0266] Implementation Scheme 10. The bispecific antibody according to Implementation Scheme 1, wherein the first antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 34, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 35, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38.

[0267] Implementation Scheme 11. The bispecific antibody according to Implementation Scheme 10, wherein the first antigen-binding portion comprises: a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 69; preferably, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69.

[0268] Implementation Scheme 12. The bispecific antibody according to any one of Implementation Schemes 1-11, wherein the second antigen-binding portion comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 94, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 95, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 96, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 97, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 98, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 99.

[0269] Implementation Scheme 13. The bispecific antibody according to any one of Implementation Schemes 1-11, wherein the second antigen-binding portion comprises: HCDR1, HCDR2 and HCDR3 in the variable region of the heavy chain as shown in SEQ ID NO: 100, and LCDR1, LCDR2 and LCDR3 in the variable region of the light chain as shown in SEQ ID NO: 101.

[0270] Implementation Scheme 14. A bispecific antibody according to any one of Implementation Schemes 1-13, wherein the second antigen-binding portion comprises a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 100, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 101.

[0271] Implementation Scheme 15. The bispecific antibody according to any one of Implementation Schemes 1-13, wherein the second antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 100, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101.

[0272] Implementation Scheme 16. A bispecific antibody according to any one of Implementation Schemes 1-15, wherein the first antigen-binding portion is murine, chimeric, or humanized.

[0273] Implementation Scheme 17. A bispecific antibody according to any one of Implementation Schemes 1-16, wherein the second antigen-binding portion is murine, chimeric, or humanized.

[0274] Implementation Scheme 18. A bispecific antibody according to any one of Implementation Schemes 1-17, wherein the first antigen-binding portion and the second antigen-binding portion are each independently Fab, scFv or scFab.

[0275] Implementation Scheme 19. The bispecific antibody according to Implementation Scheme 18, wherein the first antigen-binding portion is Fab and the second antigen-binding portion is scFv.

[0276] Implementation Scheme 20. The bispecific antibody according to any one of Implementation Schemes 1-19, wherein the bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.

[0277] Implementation Scheme 21. The bispecific antibody according to Implementation Scheme 20, wherein the first antigen-binding portion is Fab, the second antigen-binding portion is scFv, the first antigen-binding portion is fused at the C-terminus of its Fab heavy chain to the N-terminus of one of the Fc peptides of the Fc domain, and the second antigen-binding portion is fused at its C-terminus to the N-terminus of the other Fc peptide of the Fc domain.

[0278] Implementation Scheme 22. The bispecific antibody according to Implementation Scheme 21, wherein the structure of the second antigen-binding portion from the N-terminus to the C-terminus is VH-VL or VL-VH; optionally, the heavy chain variable region of the second antigen-binding portion is fused to the light chain variable region of the second antigen-binding portion via a peptide linker, preferably, the peptide linker is (GGGGS)3.

[0279] Implementation Scheme 23. The bispecific antibody according to any one of Implementation Schemes 20-22, wherein the Fc domain is an IgG Fc domain, preferably an IgG1 Fc domain.

[0280] Implementation Scheme 24. The bispecific antibody according to Implementation Scheme 23, wherein the IgG Fc domain is a human IgG Fc domain, preferably a human IgG1 Fc domain.

[0281] Implementation Scheme 25. A bispecific antibody according to any one of Implementation Schemes 20-24, wherein the Fc domain comprises an amino acid substitution that promotes association between two Fc polypeptides of the Fc domain.

[0282] Implementation Scheme 26. The bispecific antibody according to Implementation Scheme 25, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions 354C and 366Y / W, and the other Fc polypeptide comprises amino acid substitutions 349C, 366S, 368A, and 407T / V.

[0283] Implementation Scheme 27. A bispecific antibody according to any one of Implementation Schemes 20-26, wherein the Fc domain comprises amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or reduce effector function.

[0284] Implementation Scheme 28. The bispecific antibody according to Implementation Scheme 27, wherein, according to the EU number, the amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or reduce effector function are at one or more positions selected from the group consisting of: 234, 235, and 329; preferably, both Fc polypeptides of the Fc domain contain amino acid substitutions 234A, 235A, and 329G, or both contain amino acid substitutions 234A, 235A, and 329A.

[0285] Implementation Scheme 29. The bispecific antibody according to any one of Implementation Schemes 20-28, wherein the Fc domain comprises an amino acid substitution that reduces or eliminates the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A.

[0286] Implementation Scheme 30. The bispecific antibody according to Implementation Scheme 29, wherein, according to the EU number, the Fc domain comprises an amino acid substitution (a) 435R or (b) 435R and 436F occurring only in one of the Fc polypeptides.

[0287] Implementation Scheme 31. A bispecific antibody according to any one of Implementation Schemes 20-24, wherein, according to the EU designation, one of the Fc polypeptides of the Fc domain comprises amino acid substitutions: L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide comprises amino acid substitutions: (a) L234A, L235A, P329A, S354C, T366W, and H435R, or (b) L234A, L235A, P329A, S354C, T366W, H435R, and Y436F.

[0288] Implementation Scheme 32. The bispecific antibody according to any one of Implementation Schemes 1-31, wherein the bispecific antibody is bivalent.

[0289] Implementation Scheme 33. The bispecific antibody according to any one of Implementation Schemes 1-32, wherein the bispecific antibody comprises three polypeptide chains, wherein: (1) one polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 84; another polypeptide chain contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 78; and a third .... The amino acid sequence shown in SEQ ID NO: 88 has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 86; or (2) one polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 82, another polypeptide chain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 82, and yet ... The amino acid sequence shown in 88 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0290] Implementation Scheme 34. A bispecific antibody comprising three polypeptide chains, wherein: (1) one polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 84, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 78, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 88; or (2) one polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 86, another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 82, and a third polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 88.

[0291] Implementation Scheme 35. An isolated nucleic acid comprising a nucleotide sequence encoding a bispecific antibody according to any one of Implementation Schemes 1-34.

[0292] Implementation Scheme 36. A vector comprising the nucleic acid according to Implementation Scheme 35.

[0293] Implementation Scheme 37. A host cell comprising the nucleic acid according to Implementation Scheme 35, or the vector according to Implementation Scheme 36.

[0294] Implementation Scheme 38. A method for preparing a bispecific antibody according to any one of Implementation Schemes 1-34, comprising culturing a host cell according to Implementation Scheme 37 to express the bispecific antibody, and isolating and purifying the bispecific antibody in the system.

[0295] Implementation Scheme 39. A pharmaceutical composition comprising a bispecific antibody according to any one of Implementation Schemes 1-34, and a pharmaceutically acceptable excipient.

[0296] Implementation Scheme 40. Use of the bispecific antibody according to any one of Implementation Schemes 1-34 or the pharmaceutical composition according to Implementation Scheme 39 in the preparation of a medicament for treating diseases expressing GPRC5D.

[0297] Implementation Scheme 41. The use according to Implementation Scheme 40, wherein the disease is an autoimmune disease or a tumor; the tumor is preferably a non-solid tumor, more preferably multiple myeloma.

[0298] Implementation Scheme 42. The use according to Implementation Scheme 40 or 41, wherein the bispecific antibody or the pharmaceutical composition is prepared with one or more additional therapeutic agents to produce the medicament.

[0299] Implementation Scheme 43. A method for treating a disease expressing GPRC5D, comprising administering to a subject in need a therapeutically effective amount of a bispecific antibody according to any one of Implementation Schemes 1-34 or a pharmaceutical composition according to Implementation Scheme 39.

[0300] Implementation Scheme 44. The method according to Implementation Scheme 43, wherein the disease is an autoimmune disease or a tumor; the tumor is preferably a non-solid tumor, more preferably multiple myeloma.

[0301] Implementation Scheme 45. A monospecific antibody or antigen-binding portion thereof that binds to GPRC5D, comprising a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 62, 63, 64, 65, 66, or 68, and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 67 or 69.

[0302] Implementation Scheme 46. The monospecific antibody or its antigen-binding portion according to Implementation Scheme 45, comprising: (1) a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; (2) a heavy chain variable region having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; The amino acid sequence shown in SEQ ID NO: 63 has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; (3) The amino acid sequence has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 64; and the amino acid sequence has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 64. The amino acid sequence shown in SEQ ID NO: 67 has a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 65; (4) a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 67;(5) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 69; or (6) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 68, and a light ...9. The amino acid sequence shown in 69 has a light chain variable region with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0303] Implementation Scheme 47. The monospecific antibody or its antigen-binding portion according to Implementation Scheme 45 or 46, comprising: (1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (3) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (4) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; or (5) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69.

[0304] Implementation Scheme 48. An antibody-drug conjugate comprising a monospecific antibody or its antigen-binding portion according to any one of Implementation Schemes 45-47.

[0305] Implementation Scheme 49. An anti-GPRC5D chimeric antigen receptor comprising a monospecific antibody or its antigen-binding portion according to any one of Implementation Schemes 45-47.

[0306] Implementation Scheme 50. An engineered immune effector cell comprising the anti-GPRC5D chimeric antigen receptor as described in Implementation Scheme 49.

[0307] For clarity, this disclosure is further illustrated by examples, but these examples are not intended to limit the scope of this disclosure. The reagents used in this disclosure are typically commercially available and can be used without further purification.

[0308] Example 1: Antigen Immunization 1. DNA antigen immunization The cDNA sequence encoding the human GPRC5D protein (amino acid sequence as shown in SEQ ID NO: 41) was obtained through gene synthesis and subcloned into the expression vector pcDNA3.1(+) to construct the plasmid pcDNA3.1-hGPRC5D. Large-scale plasmid preparation was performed according to the instructions of the endotoxin-free large-scale extraction kit (QIAGEN, catalog number: 12391).

[0309] Using plasmid pcDNA3.1-hGPRC5D as the antigen, A / J mice (Nanjing University Model Animal Institute), BALB / c mice (Shanghai Lingchang), and SJL mice (Beijing Vital River) were immunized, respectively. First, hyaluronidase (Sigma, catalog number: H4272) was pre-injected into the muscles of the left and right hind limbs of each mouse for pretreatment. Then, CpG (InvivoGen, catalog number: tlrl-1826) and plasmid pcDNA3.1-hGPRC5D were mixed at a 1:1 mass ratio, and the mixed antigen complex was injected into the pretreated muscle sites of the mice using a live gene delivery system (Shanghai Tarissa Biotechnology Co., Ltd., type II). The same immunization was repeated every 2-3 weeks, for a total of 4 immunizations. After immunization, serum was collected from each mouse.

[0310] 2. Evaluation of mouse serum titers To evaluate the immune response to anti-human GPRC5D antibodies produced in mice immunized with the DNA antigen, antibody titers in mouse serum were detected using FACS. Mice with higher serum titers were selected for subsequent pre-splenic cell fusion pulse immunization.

[0311] 3. Cell antigen immunity Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, catalog number: L3000015), plasmid pcDNA3.1-hGPRC5D was transfected into CHO-K1 cells to obtain a stable, high-expression hGPRC5D cell line, CHO-K1-hGPRC5D. hi The above-mentioned CHO-K1-hGPRC5D is used. hiCells were used as antigens for shock immunization in mice: 3-4 days before spleen cell fusion, the final cell concentration was 5 × 10⁻⁶. 7 CHO-K1-hGPRC5D per mL hi The cell suspension was mixed with CpG (InvivoGen, catalog number: tlrl-1826) at a final concentration of 0.5 mg / mL and injected into mice intraperitoneally at a dose of 100 µL / mouse.

[0312] Example 2: Screening of mouse anti-human GPRC5D antibodies 1. Preparation of hybridoma cells On the day of fusion, mouse spleens were aseptically harvested, ground, and then lysed with erythrocyte lysis buffer (Sigma, catalog number: R7757). After washing the cells with PBS, the spleen cells were resuspended in electrofusion buffer (BTX, catalog number: 47-0001). The spleen cells were then mixed with SP2 / 0 mouse myeloma cells at a cell ratio of 2:1, and fusion was performed using an electrofusion apparatus (BTX). The fused cells were diluted with hybridoma medium (Gibco, catalog number: 12045-076) containing 1 × HAT (Gibco, catalog number: 21060-017) and cultured at 37°C and 5% CO2 for 7-10 days to prepare hybridoma cells. The culture supernatant of the hybridoma cells was collected for screening of mouse anti-human GPRC5D antibodies.

[0313] 2. Construction of stable cell lines To evaluate the species-cross-binding activity of mouse anti-human GPRC5D antibodies at the cellular level, the following cell lines were constructed: cDNA encoding full-length monkey GPRC5D (amino acid sequence as shown in SEQ ID NO: 42) and mouse GPRC5D (amino acid sequence as shown in SEQ ID NO: 43) were synthesized via gene synthesis and then subcloned into the vector pcDNA3.1(+) to obtain recombinant plasmids pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D. Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, catalog number: L3000015), plasmids pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D were transfected into CHO-K1 cells to obtain stable CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cell lines, respectively.

[0314] 3. Initial screening using cell ELISA Collect CHO-K1-hGPRC5D in the logarithmic growth phase hiCells were resuspended in DMEM / F-12 medium (Gibco, catalog number: 11320033) containing 10% FBS and the cell concentration was adjusted to 1 × 10⁻⁶. 6 Hybridoma cell culture supernatant was seeded at a rate of 100 µL / well in 96-well plates and incubated overnight at 37°C and 5% CO2. The next day, the plates were washed with PBS, and 4% (v / v) paraformaldehyde (Beyotime, catalog number: P0099) was added for fixation at room temperature for 30 minutes. After washing with PBS, 200 µL / well of PBS containing 3% (v / v) BSA was added, and the plates were blocked at room temperature for 2 hours. After blocking, 100 µL / well of hybridoma cell culture supernatant was added, and the plates were incubated at room temperature for 2 hours; after washing with PBS, HRP-conjugated goat anti-mouse IgG+IgM(H+L) antibody (Jackson Immuno Research, catalog number: 115-035-068) was added, and the plates were incubated at room temperature for 1 hour. After washing with PBS, add 100 µL / well of TMB reaction solution (Solepro, catalog number: RP1200), incubate at room temperature in the dark for 5 minutes, and terminate the reaction with 0.5 M H2SO4. Read OD values ​​using a Bio-rad iMark microplate reader. 450 nm Absorbance. Hybridoma cells corresponding to culture supernatants with absorbance greater than 1.0 were selected as positive clones for subsequent FACS screening.

[0315] 4. Flow cytometry (FACS) for antibody screening The cell concentration was 5 × 10⁻⁶. 5 Cell suspension of CHO-K1-cynoGPRC5D cells at a density of 100 µL / well was seeded into 96-well U-plates. 100 µL of culture supernatant from positive clones was added to each well, and the mixture was incubated at 4°C for 1 hour. After washing with PBS containing 2% (v / v) FBS, Alexa Fluor 488 conjugated goat anti-mouse IgG+IgM(H+L) antibody (Jackson Immuno Research, catalog number: 115-545-044) was added, and the cells were incubated at 4°C for 1 hour. Cells were washed and resuspended in PBS containing 2% (v / v) FBS, and fluorescence signals were detected using flow cytometry (Sartorius, iQue3). The binding of anti-hGPRC5D antibody to CHO-K1-cynoGPRC5D cells was analyzed by mean fluorescence intensity (MFI).

[0316] 5. Subcloning preparation Selected cells after ELISA and FACS screening, and then compared with CHO-K1-hGPRC5D hiHybridoma cells that were positive for both CHO-K1-cynoGPRC5D were expanded and cultured. The expanded cells were mixed with semi-solid medium D (Stemcell, catalog number: 3810) at a volume ratio of 1:10, and then incubated at 37°C and 5% CO2 for 5 days. Single-clonal cell clusters were aspirated under a 4x microscope and transferred to 96-well plates coated with hybridoma medium (Gibco, catalog number: 12045-076), and cultured for another 2 days at 37°C and 5% CO2. The supernatant from each subclonal cell culture was collected for further subclonal screening.

[0317] 6. Subcloning screening (1) FACS screening of anti-hGPRC5D antibodies The cell concentration was 3 × 10⁻⁶. 5 NCI-H929 cell suspension was seeded at 100 µL / well in 96-well U-plates, and 100 µL / well of subclonal cell culture supernatant or an equal volume of culture medium was added. The mixture was then incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (v / v) FBS, Alexa Fluor 488 conjugated goat anti-mouse IgG+IgM(H+L) antibody (Jackson Immuno Research, catalog number: 115-545-044) was added, and the cells were incubated at 4°C for 1 hour. The cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3). The binding of mouse anti-hGPRC5D antibody to NCI-H929 cells was analyzed by the mean fluorescence intensity (MFI). Some results are shown in Table 1.

[0318] Table 1. FACS binding assay of some mouse anti-hGPRC5D antibodies to NCI-H929 cells

[0319] (2) Cellular ELISA screening of anti-hGPRC5D antibody Collect CHO-K1-hGPRC5D in the logarithmic growth phase. hi Cells, CHO-K1-cynoGPRC5D cells, CHO-K1-murineGPRC5D cells, and CHO-K1 cells were resuspended in DMEM / F-12 (Gibco, catalog number: 11320033) medium containing 10% (v / v) FBS, and the cell concentration was adjusted to 1 × 10⁻⁶. 6Cells were cultured at a density of 100 µL / well in 96-well plates and incubated overnight at 37°C with 5% CO2. The next day, the plates were washed with PBS, and 4% (v / v) paraformaldehyde (Beyotime, catalog number: P0099) was added for fixation at room temperature for 30 minutes. After washing with PBS, 200 µL / well of PBS containing 3% (v / v) BSA was added, and the plates were blocked at room temperature for 2 hours. After blocking, 50 µL / well of subcloned cell culture supernatant was added to each well, and the plates were incubated at room temperature for 2 hours. After washing with PBS, HRP-conjugated goat anti-mouse IgG+IgM(H+L) antibody (Jackson Immuno Research, catalog number: 115-035-068) was added, and the plates were incubated at room temperature for 1 hour. After washing with PBS, add 100 µL / well of TMB reaction solution (Solepro, catalog number: RP1200), incubate at room temperature in the dark for 5 minutes, and terminate the reaction with 0.5 M H2SO4. Read OD values ​​using a Bio-rad iMark microplate reader. 450 nm Absorbance value. For example... Figure 1 As shown, typical mouse anti-hGPRC5D antibodies (e.g., mu-89, mu-105, mu-126, mu-128, mu-131, mu-153, mu-164, mu-169, mu-180, and mu-183) target CHO-K1-hGPRC5D. hi The absorbance values ​​of the cells, CHO-K1-cynoGPRC5D cells and / or CHO-K1-murineGPRC5D cells, were at least 2-fold different from those of the control cells CHO-K1.

[0320] Example 3: Preparation of anti-hGPRC5D chimeric antibody 1. Sequencing of the variable region encoding mouse anti-hGPRC5D antibody Total RNA was isolated from the selected hybridoma cells according to the instructions of the RNA extraction kit (Takara, catalog number: 9767), and first-strand cDNA was synthesized using a reverse transcription kit (Thermo, catalog number: K1652). Using the first-strand cDNA as a template, primers for the mouse heavy and light chain constant regions were mixed, and the variable region sequence of the mouse anti-hGPRC5D antibody was obtained by cloning and sequencing using polymerase chain reaction (PCR).

[0321] 2. Construction of chimeric antibody expression vector Using a chemical synthesis method, the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of mouse anti-hGPRC5D antibody were linked to the nucleotide sequences of the heavy chain constant region and light chain constant region of human IgG1, respectively. Recombinant human-mouse chimeric antibody expression vectors were constructed using the pcDNA3.1(+) vector and transfected for expression to prepare chimeric antibodies. The VH / VL amino acid sequences and full-length amino acid sequences of each chimeric antibody are shown in Table 2.

[0322] Table 2. Chimeric antibody VH / VL and full-length amino acid sequences (numbers in the table indicate sequence numbers SEQ ID NO:)

[0323] 3. Construction of positive control antibody expression vector GC5B596 was selected as the reference antibody (monoclonal antibody, source WO2018017786A2). The nucleotide sequences of antibodies VH (SEQ ID NO: 44) and VL (SEQ ID NO: 45) were obtained through chemical synthesis and ligated to the nucleotide sequences of the constant region of the human IgG1 heavy chain and the constant region of the Kappa light chain, respectively. An expression vector for expressing the positive reference antibody was constructed using the pcDNA3.1(+) vector and transfected for expression to prepare the positive reference antibody. The positive reference antibody (monoclonal antibody) was named BM-mAb in this paper.

[0324] 4. Preparation of anti-hGPRC5D chimeric antibody and positive reference antibody Transient transfection expression of anti-hGPRC5D antibody was performed according to the operating manual of the expiCHO expression system (Gibco, catalog number: A29129). After transfection, expiCHO cells were cultured at 37°C with 8% CO2 for 7 days with shaking. The cell culture supernatant was collected, and the clarified culture supernatant was then loaded onto a protein A column (GE Healthcare, catalog number: 17-5474). The protein A column was washed with 10 column volumes of PBS buffer, and then eluted and collected with acetate buffer (300 mM acetic acid, pH 3.6). The collected IgG antibody fraction was then replaced with PBS buffer through an ultrafiltration device (molecular weight cutoff 30 kDa, Millipore, catalog number: UFC903024) to obtain the anti-hGPRC5D antibody solution.

[0325] Example 4: Assay of the binding activity of anti-hGPRC5D antibody to cells stably expressing human GPRC5D 1. Binding properties of anti-hGPRC5D chimeric antibody to cells stably overexpressing human GPRC5D The cell concentration was 3 × 10⁻⁶. 5CHO-K1-hGPRC5D per mL hi (GPRC5D expression abundance is approximately 2E+06 ~ 3E+06 antigens / cell) Cell suspension was seeded at 100 µL / well in 96-well U-plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS, and then PE-conjugated goat anti-human IgG Fcγ antibody (Jackson ImmunoResearch, catalog number: 109-116-170) was added and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3). The mean fluorescence intensity (MFI) of the staining was used to analyze the interaction between anti-hGPRC5D antibody and CHO-K1-hGPRC5D. hi Cell binding. Calculating EC. 50 The analysis results are as follows Figure 2 As shown, the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169 and Chi-180 all exhibited concentration gradient-dependent binding activity on hGPRC5D cells with high expression, and had a higher maximum antigen binding amount than BM-mAb.

[0326] 2. Binding properties of anti-hGPRC5D chimeric antibody to cells with stable low expression of human GPRC5D Following the instructions for the Lipofectamin 3000 transfection reagent (Thermo, catalog number: L3000015), plasmid pcDNA3.1-hGPRC5D was transfected into CHO-K1 cells to obtain CHO-K1-hGPRC5D. low Stable low-expression cell lines (GPRC5D expression abundance of approximately 2000-2500 antigens / cell). Cell concentration was set at 3 × 10⁻⁶ cells / cell. 5 CHO-K1-hGPRC5D per mL lowCell suspension was seeded at 100 µL / well in 96-well U-type plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold serial dilution) was added, and the mixture was incubated at 4°C for 1 hour. After washing cells with PBS containing 2% (v / v) FBS, PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number: 109-116-170) was added, and the cells were incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3). The mean fluorescence intensity (MFI) of the staining was used to analyze the interaction between anti-hGPRC5D antibody and CHO-K1-hGPRC5D. low Cellular binding. For example... Figure 3 As shown, on cells with low hGPRC5D expression, the chimeric antibodies Chi-89, Chi-105, Chi-164, and Chi-180 had comparable binding levels to BM-mAb, while Chi-128, Chi-131, and Chi-169 exhibited stronger target binding ability.

[0327] Example 5: Assay of the binding activity of anti-hGPRC5D antibody to tumor cells Cell concentrations of 3 × 10⁻⁶ were respectively... 5 NCI-H929 and MM.1S human myeloma cell suspensions were seeded at 100 µL / well in 96-well U-plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-1000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (v / v) FBS, PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number: 109-116-170) was added, and the cells were incubated at 4°C for 1 hour. The cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3), and the binding of anti-hGPRC5D antibody to human myeloma cells was analyzed by mean fluorescence intensity (MFI). Figure 4A and Figure 4B It was found that the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131 and Chi-169 all exhibited concentration gradient-dependent binding activity on NCI-H929 and MM.1S cells.

[0328] Example 6: Determination of cross-species activity of anti-hGPRC5D antibody Cell concentrations of 5 × 10⁻⁶ were respectively... 5CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cell suspensions were seeded at 100 µL / well in 96-well U-plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.6-10000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. After washing with PBS containing 2% (v / v) FBS, PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number: 109-116-170) was added, and incubated at 4°C for 1 hour. Cells were washed and resuspended with PBS containing 2% (v / v) FBS, and fluorescence signals were detected using flow cytometry (Sartorius, iQue3). The binding of anti-hGPRC5D antibody to CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells was analyzed by mean fluorescence intensity (MFI). Figure 5A and 5B As shown, chimeric antibodies Chi-89, Chi-128, Chi-131, and Chi-169 exhibited concentration-gradient binding activity in both CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells, indicating that these anti-hGPRC5D antibodies, compared to BM-mAb, possess cross-binding activity with both monkey and mouse GPRC5D. In contrast, chimeric antibody Chi-105 only exhibits cross-binding activity with monkey GPRC5D.

[0329] Example 7: Specificity verification of anti-hGPRC5D antibody 1. Verification of the binding of anti-hGPRC5D chimeric antibody to its family of proteins at the cellular level. Besides GPRC5D, the GPRC family of proteins includes GPRC5A (RAIG1), GPRC5B (RAIG2), and GPRC5C (RAIG3). cDNAs encoding full-length GPRC5A (SEQ ID NO: 46), GPRC5B (SEQ ID NO: 47), and GPRC5C (SEQ ID NO: 48) were synthesized and subcloned into the vector pcDNA3.1(+) to obtain recombinant plasmids pcDNA3.1-GPRC5A-GFP, pcDNA3.1-GPRC5B-GFP, and pcDNA3.1-GPRC5C-GFP. Following the instructions of the Lipofectamin 3000 transfection reagent (Thermo, catalog number: L3000015), the recombinant plasmid DNA was transfected into CHO-K1 cells to obtain stable cell lines CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C.

[0330] Cell concentrations of 5 × 10⁻⁶ were respectively... 5 CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cell suspensions were seeded at 100 µL / well in 96-well U-plates. Serially diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. After washing with PBS containing 2% (v / v) FBS, APC-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number: 109-135-098) was added, and incubated at 4°C for 1 hour. Cells were washed and resuspended in PBS containing 2% (v / v) FBS. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3). The binding of anti-hGPRC5D antibody to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells was analyzed by mean fluorescence intensity (MFI) of the staining. Figure 6A , 6B As shown in Figure 6C, the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, and Chi-169 did not bind to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells at concentrations ranging from 4.1 to 3000 ng / mL, while BM-mAb showed nonspecific binding to CHO-K1-GPRC5A cells at concentrations greater than 1000 ng / mL.

[0331] 2. Validation of the binding of anti-hGPRC5D chimeric antibody to CHO-K1 cells First, following the instructions of the EZ-linkNHS-Biotin reagent (Thermo, catalog number: 20217), biotin-conjugated anti-hGPRC5D antibodies were prepared and named Biotin-BM-mAb, Biotin-Chi-89, Biotin-Chi-128, and Biotin-Chi-131, respectively, where Biotin represents biotin.

[0332] The cell concentration was 3 × 10⁻⁶. 5CHO-K1 cell suspension (cells / mL) was seeded at 100 µL / well in 96-well U-plates. Serially diluted biotin-conjugated anti-hGPRC5D antibody (3-fold serial dilution, final concentration range 4.6-10000 ng / mL) was added, mixed, and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS, and then PE-conjugated streptavidin (BD, catalog number: 554061) was added, followed by incubation at 4°C for 1 hour. Cells were washed with PBS containing 2% (v / v) FBS and resuspended. Fluorescence signals were detected using flow cytometry (Sartorius, iQue3). The mean fluorescence intensity (MFI) was used to analyze whether the biotin-conjugated anti-hGPRC5D antibody, with its amplified MFI signal, exhibited non-specific binding to CHO-K1 cells. Results are as follows: Figure 7 As shown, the biotin-conjugated chimeric antibodies Chi-89, Chi-128, and Chi-131 all produced non-binding signals with CHO-K1 cells.

[0333] Example 8: Construction, expression, and purification of anti-GPRC5D humanized antibody The chimeric antibodies Chi-131 and Chi-128 against GPRC5D were humanized. Four humanized antibodies were obtained from the humanization of Chi-131, named hu131-v1, hu131-v2, hu131-v3, and hu131-v4; one humanized antibody was obtained from the humanization of Chi-128, named hu128-v1. The amino acid and nucleotide sequences of the heavy and light chains of each humanized antibody are shown in Table 3.

[0334] Table 3. Sequence information of humanized antibodies (SEQ ID NO:)

[0335] Nucleotide sequences encoding the aforementioned humanized antibodies were synthesized and cloned into pcDNA3.1(+) expression vectors. Each antibody was expressed using the ExpiCHO expression kit (Thermo Fisher, catalog number A29133). First, the constructed expression vectors containing the aforementioned nucleotide sequences were transfected into ExpiCHO cells (CHO-S, Thermo Fisher Scientific). The cells were then cultured in ExpiCHO expression medium at 37°C in a humidified atmosphere containing 8% CO2 on a tracked shaker platform rotating at 130 rpm. The culture supernatant was collected, and the protein was purified using protein A magnetic beads (Genscript, catalog number L00273). Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).

[0336] Example 9: Assay of the binding activity of anti-GPRC5D chimeric antibody and humanized antibody The binding activity of chimeric antibodies Chi-131 and Chi-128 and their humanized antibodies to human GPRC5D was determined by binding to NCI-H929 cells (source: Nanjing Kebai Biotechnology).

[0337] The cell concentration was 1 × 10⁻⁶ 6 NCI-H929 human myeloma cell suspension (cells / mL) was seeded at 100 µL / well in 96-well U-plates. Serially diluted anti-GPRC5D antibody (initial concentration range 310-1586 nM, 5-fold serial dilution) was added, mixed, and incubated at 4°C for 1 hour. After washing the cells with pre-chilled running buffer (MACS, catalog number 130-091-221), PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number 109-116-170) was added, and the cells were incubated at 4°C for 0.5 hours. The cells were washed with pre-chilled running buffer and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3), and the binding of anti-hGPRC5D antibody to human myeloma cells was analyzed by mean fluorescence intensity (MFI). The binding activity of humanized antibodies (hu131-v1, hu131-v2, hu131-v3, hu131-v4, hu128-v1) and chimeric antibodies (Chi-131, Chi-128) to the GPRC5D target antigen of NCI-H929 cells is as follows: Figure 8 As shown, humanized antibodies (hu131-v1, hu131-v2, hu131-v3, hu131-v4, hu128-v1) and chimeric antibodies (Chi-131, Chi-128) all exhibited concentration-gradient-dependent binding activity on NCI-H929 cells.

[0338] Example 10: Construction, expression, and purification of anti-GPRC5D / anti-CD3 bispecific antibody (1) Anti-GPRC5D / anti-CD3 bispecific antibodies GC35 and GC39 according to Figure 9 The configurations shown are used to construct anti-GPRC5D / anti-CD3 bispecific antibodies GC35 and GC39, where the first antigen-binding moiety is the Fab that binds to GPRC5D, and the second antigen-binding moiety is the scFv that binds to CD3. Specifically, GC35 and GC39 have three polypeptide chains, and the amino acid and nucleotide sequences of the three polypeptide chains are shown in Table 4-1.

[0339] Nucleotide sequences encoding anti-GPRC5D-HC, anti-GPRC5D-LC, and anti-CD3-scFv-Fc were synthesized and cloned into pcDNA3.1(+) expression vectors, respectively. Using the ExpiCHO transfection kit (Thermo Fisher, catalog number A29133), the constructed expression vectors were co-transfected into ExpiCHO cells at a ratio of anti-GPRC5D-HC: anti-GPRC5D-LC: anti-CD3-scFv-Fc = 1:1.5:1.5, with a transfection density of 6 × 10⁶ cells / year. 6 Cells / mL. Cell culture supernatant was collected by centrifugation on days 10-15 post-transfection. Protein purification was performed using the ÄKTA pure protein purification system (GE Healthcare) via protein A affinity chromatography, ion exchange chromatography, and gel chromatography. Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific). Electrophoresis (reducing SDS-PAGE), time-of-flight MS, and sequence analysis confirmed the acquisition of the aforementioned bispecific antibody with the expected structure and sequence.

[0340] (2) Reference product Talquetamab The anti-GPRC5D / anti-CD3 bispecific antibody Talquetamab was constructed as a benchmark (BM). Specifically, Talquetamab has four polypeptide chains, and the amino acid sequences are shown in Table 4-2. The nucleotide sequences encoding each polypeptide chain of Talquetamab were synthesized and cloned into pcDNA3.1(+) expression vectors, respectively. Using the ExpiCHO transfection kit (Thermo Fisher, A29133), Talquetamab anti-GPRC5D-HC and anti-GPRC5D-LC were co-transfected into the first ExpiCHO sample at a ratio of anti-GPRC5D-HC: anti-GPRC5D-LC = 1:1.5 to express the anti-GPRC5D monoclonal antibody; Talquetamab anti-CD3-HC and anti-CD3-LC were co-transfected into the second ExpiCHO sample at a ratio of anti-CD3-HC: anti-CD3-LC = 1:1.5 to express the anti-CD3 monoclonal antibody. The transfection density of both ExpiCHO samples was 6 × 10⁻⁶. 6 Cells were cultured continuously for 10-15 days post-transfection, and the supernatant was collected by centrifugation.

[0341] The obtained supernatant was purified using an ÄKTA pure protein purification system (GE Healthcare). The purified anti-GPRC5D and anti-CD3 monoclonal antibodies were assembled in vitro in equimolar amounts to obtain Talquetamab. Specifically, the purified antibody solution was replaced with PBS (pH 7.4) and concentrated. 2 mg of each monoclonal antibody was taken, resulting in a total antibody volume of 4 mg (the molar ratio of anti-GPRC5D to anti-CD3 monoclonal antibody was approximately 1:1), and the volume was brought to 0.9 mL with PBS. 6 mg of 2-mercaptoethylamine•HCl (2-MEA, final concentration 50 mM) was dissolved in 100 μL of PBS and rapidly added to the prepared antibody solution to prepare a 1 mL reaction system. The mixture was thoroughly mixed, incubated at 37°C for 90 minutes, cooled to room temperature, and concentrated by ultrafiltration to remove 2-MEA. The solution was then stored in PBS. After overnight oxidation at 4°C, the antibody concentration was measured.

[0342] Table 4-1. Sequence information of anti-GPRC5D / anti-CD3 bispecific antibodies

[0343] Table 4-2. Serial information of the reference sample

[0344] Example 11: The interaction between anti-GPRC5D / anti-CD3 bispecific antibody and multiple myeloma cells expressing GPRC5D combine The binding of anti-GPRC5D / anti-CD3 bispecific antibodies (including Talquetamab, GC35, and GC39) to GPRC5D-expressing RPMI-8226 cells (source: Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), MM.1S cells (source: Beina Biotechnology), and NCI-H929 cells (source: Nanjing Kebai Biotechnology) was analyzed by flow cytometry. The negative control was hIgG1 monoclonal antibody (Baiying Biotechnology, catalog number B117901).

[0345] RPMI-8226 cells, MM.1S cells, and NCI-H929 cells in logarithmic growth phase were adjusted to a cell density of 1 × 10⁻⁶ using RPMI-1640 medium (Hyclone, catalog number SH30809.01) containing 2% (v / v) FBS (fetal bovine serum). 6 ~2 × 10 6Cells were seeded at a concentration of 50 μL / well in a 96-well U-shaped cell culture plate (Costar, catalog number: 3799). Different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody were prepared using the above-mentioned culture medium, with a maximum antibody concentration of 200 nM, using a 5-fold serial dilution for a total of 9 concentration gradients. 50 μL of each antibody concentration was added to each well of the 96-well cell culture plate, mixed, and incubated at 4°C for 1 hour. After washing the cells with pre-chilled Running buffer (MACS, catalog number 130-091-221) and discarding the supernatant, 100 μL of pre-chilled PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number 109-116-170) was added to each well for resuspending, mixed, and incubated at 4°C for 30 minutes. After washing, cells were resuspended in 100 μL of pre-cooled running buffer per well, mixed well, and then collected on a flow cytometer (Sartorius, iQue3) for data analysis.

[0346] Table 5 and Figures 10A-10C The binding ability of the anti-GPRC5D / anti-CD3 bispecific antibody to RPMI-8226, MM.1S, and NCI-H929 cells was demonstrated. Results showed that GC35 and GC39 exhibited stronger binding affinity to the target cells RPMI-8226, MM.1S, and NCI-H929 than Talquetamab.

[0347] Table 5. Binding of anti-GPRC5D / anti-CD3 bispecific antibody to target cells EC 50 Value and maximum combined value

[0348] The " / " indicates that the curve cannot be fitted to a numerical value.

[0349] Example 12: Binding of anti-GPRC5D / anti-CD3 bispecific antibody to CD3-expressing T cells The binding of anti-GPRC5D / anti-CD3 bispecific antibodies (including GC35, GC39, and Talquetamab) to CD3 on the surface of T cells was analyzed by flow cytometry. The negative control was hIgG1 monoclonal antibody (Baiying Biotechnology, catalog number B117901).

[0350] Human PBMCs (peripheral blood mononuclear cells) cells were subjected to CD3 magnetic beads (Miltenyi, catalog number 130-097-043) for CD3 PCR. + T cell sorting. Frozen PBMCs were thawed, centrifuged and washed twice, and then counted. Cells were sorted at 10... 7Add running buffer (MACS, catalog number 130-091-221) at a ratio of 80 μL per cell, and then add it at a ratio of 10 μL per 10 cells. 7 Add CD3 magnetic beads at a ratio of 20 μL per cell, mix well, and incubate at 4°C for 15 minutes. Repeat the process for every 10 cells. 7 Add 1-2 mL of running buffer per cell to wash the cells. After centrifugation and discarding the supernatant, resuspend the cell pellet in 500 μL of running buffer. Place the LS sorting column (Miltenyi, catalog number 130-042-401) on the MidiMACS Starting Kit (LS) magnetic rack (Miltenyi, catalog number 130-091-051), rinse, add the cell suspension, and wash three times. Remove the LS sorting column from the magnetic rack and place it on a clean centrifuge tube. Add 5 mL of buffer to collect the sorted CD3+. + T cells.

[0351] After counting, CD3 + T cells use running buffer to adjust cell density to 1. × 10 6 ~ 2 × 10 6 Cells were seeded at a concentration of 50 μL / well in a 96-well U-shaped cell culture plate (Costar, catalog number: 3799). Different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody were prepared using running buffer, with a maximum concentration of 200 nM, using 5-fold serial dilutions for a total of 10 concentration gradients. 50 μL of each antibody concentration was added to the 96-well cell culture plate, mixed, and incubated at 4°C for 1 hour. After washing the cells with pre-chilled running buffer (MACS, catalog number 130-091-221) and discarding the supernatant, 100 μL of pre-chilled PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number 109-116-170) was added to each well for resuspending, mixed, and incubated at 4°C for 30 minutes. After washing, cells were resuspended in 100 μL of pre-cooled running buffer per well, mixed well, and then collected on a flow cytometer (Sartorius, iQue3) for data analysis.

[0352] Table 6 and Figure 11 The anti-GPRC5D / anti-CD3 bispecific antibody showed activity against CD3. + The binding capacity of T cells was shown to be that GC35 and GC39 could bind to CD3. + T cells.

[0353] Table 6. Anti-GPRC5D / anti-CD3 bispecific antibody and CD3 + T-cell binding EC 50 Value and maximum combined value

[0354] Example 13: Killing of multiple myeloma target cells by anti-GPRC5D / anti-CD3 bispecific antibody This study investigated the cytotoxic effects of anti-GPRC5D / anti-CD3 bispecific antibodies (including GC35, GC39, and Talquetamab) on target cells NCI-H929 (source: Nanjing Kebai Biotechnology), MM.1S (source: Beina Biotechnology), and RPMI-8226 (source: Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) using human PBMCs to provide T cells. The negative control was hIgG1 monoclonal antibody (Baiying Biotechnology, catalog number B117901).

[0355] Target cells were adjusted to a density of 5 × 10⁶ cells using RPMI-1640 medium (Hyclone, catalog number SH30809.01) containing 2% (v / v) FBS. 5 50 μL of anti-GPRC5D / anti-CD3 bispecific antibody was seeded per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody were prepared using the above-mentioned medium, with a maximum antibody concentration of 10 nM. For NCI-H929 and MM.1S cells, nine concentration gradients were prepared by 5-fold serial dilution; for RPMI-8226 cells, eight concentration gradients were prepared by 6-fold serial dilution. 50 μL of the antibody at different concentrations was added to each well of the 96-well cell culture plate. Human PBMCs, as effector cells, were adjusted to a cell density of 2.5 × 10⁶ cells using the same medium. 6 Cells / mL, 100 μL per well. The following groups were set up: drug administration group (50 μL target cells + 100 μL effector cells + 50 μL antibody), target cell group (50 μL target cells + 150 μL culture medium), effector cell group (100 μL human PBMCs + 100 μL culture medium), target cell + effector cell group (50 μL target cells + 100 μL effector cells + 50 μL culture medium), blank control group (200 μL culture medium), lysis buffer control group (200 μL culture medium + 20 μL lysis buffer), and target cell maximum release group (50 μL target cells + 150 μL culture medium + 20 μL lysis buffer). The effector-to-target ratio was 10:1, and all cells were incubated for 24 hours. The lysis buffer (Promega, catalog number G182A) in the target cell maximum release group and lysis buffer control group was added 45 min before the assay. CytoTox96 was used. ®The non-radioactive cytotoxicity assay kit (Promega, G1780) was used for detection, and the absorbance value at a wavelength of 490 nm was measured in an ELISA reader to calculate the cell lysis rate.

[0356]

[0357] Figures 12A-12C Table 7 shows the lysis rates of various tumor cells against the anti-GPRC5D / anti-CD3 bispecific antibody. The anti-GPRC5D / anti-CD3 bispecific antibody GC35 showed superior killing activity against NCI-H929, MM.1S, and RPMI-8226 cells compared to GC39 and Talquetamab.

[0358] Table 7. Lysis EC5 of each antibody on various tumor cells 50 Value and maximum pyrolysis rate

[0359] Example 14: T cell activation induced by anti-GPRC5D / anti-CD3 bispecific antibody This study investigated the activation of T cells by anti-GPRC5D / anti-CD3 bispecific antibodies (including GC35, GC39, and Talquetamab) in the presence of myeloma target cells by detecting the expression levels of T cell activation markers CD69 and CD25. The activation capacity of T cells was determined by the concentration-dependent upregulation of the markers. The negative control was hIgG1 monoclonal antibody (Baiying Biotechnology, catalog number B117901).

[0360] Target cells MM.1S (source: Beina Biotechnology) were adjusted to a density of 5 × 10⁶ cells using RPMI-1640 medium (Hyclone, catalog number SH30809.01) containing 2% (v / v) FBS. 5 50 μL of anti-GPRC5D / anti-CD3 bispecific antibody was seeded per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody were prepared using the above-mentioned culture medium, with a maximum antibody concentration of 10 nM, serially diluted 5-fold for a total of 8 concentration gradients. 50 μL of each antibody concentration was added to the 96-well cell culture plate. Human PBMCs, the effector cells, were adjusted to a cell density of 2.5 × 10⁶ cells / mL using experimental culture medium. 6Cells / mL, 100 μL per well. The following groups were set up: drug administration group (50 μL target cells + 100 μL effector cells + 50 μL antibody), target cell group (50 μL target cells + 150 μL culture medium), effector cell group (100 μL human PBMCs + 100 μL culture medium), target cell + effector cell group (50 μL target cells + 100 μL effector cells + 50 μL culture medium), and blank control group (200 μL culture medium). The effector-to-target ratio was 10:1, and the cells were incubated for 24 hours. Centrifuge to remove supernatant, add pre-chilled running buffer (MACS, catalog number 130-091-221), centrifuge to wash cells, remove supernatant, add 100 μL running buffer to each well, mix cells, add 2.5 μL each of CD4-BV421 (BD, catalog number 564713) and CD8-PE-Cy7 (BD, catalog number 557746) and 10 μL each of CD25-APC (BD, catalog number 555434) and CD69-FITC (BD, catalog number 555530) to each well, incubate on ice for 30 minutes. Wash cells with pre-chilled running buffer, resuspend cells in 100 μL / well running buffer, mix well, and then place in a flow cytometer for analysis.

[0361] Figures 13A-13D Table 8 shows the degree of T cell activation in the presence of MM.1S target cells, where CD25 / CD4 represents CD25 + Cell number as a percentage of CD4 + The ratio of cell number, CD69 / CD4 indicates CD69 + Cell number as a percentage of CD4 + The ratio of cell numbers, CD25 / CD8, represents CD25 / CD8. + Cell count as a percentage of CD8 + The ratio of cell numbers, CD69 / CD8, indicates CD69 + Cell count as a percentage of CD8 + The proportion of cell numbers. The expression of CD25 and CD69 was significantly increased compared to the negative control, indicating that T cells were activated; both GC35 and GC39 can effectively activate T cells.

[0362] Table 8. Effects of anti-GPRC5D / anti-CD3 bispecific antibodies on T cell activation in the presence of MM.1S in ECMO cells. 50 value

[0363] Example 15: Cytokine release induced by anti-GPRC5D / anti-CD3 bispecific antibody This study investigated the cytokine release induced by anti-GPRC5D / anti-CD3 bispecific antibodies (including GC35 and Talquetamab) in the presence of target cells by detecting the levels of IL-2, IL-6, TNF-α, and IFN-γ. The safety of the bispecific antibodies was assessed based on the amount of cytokine released.

[0364] Target cells NCI-H929 (source: Nanjing Kebai Biotechnology) were adjusted to a density of 5 × 10⁶ cells using RPMI-1640 medium (Hyclone, catalog number SH30809.01) containing 2% (v / v) FBS. 5 50 μL of anti-GPRC5D / anti-CD3 bispecific antibody was seeded per well in a 96-well cell culture plate (Eppendorf, catalog number: 0030730199). Different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody were prepared using the above-mentioned culture medium, with a maximum antibody concentration of 10 nM, serially diluted 5-fold for a total of 9 concentration gradients. 50 μL of each antibody concentration was added to the 96-well cell culture plate. Human PBMCs, as effector cells, were adjusted to a cell density of 2.5 × 10⁻⁶ cells / mL using experimental culture medium. 6 Cells / mL, 100 μL per well. The following groups were set up: drug administration group (50 μL target cells + 100 μL effector cells + 50 μL antibody), target cell group (50 μL target cells + 150 μL culture medium), effector cell group (100 μL human PBMCs + 100 μL culture medium), target cell + effector cell group (50 μL target cells + 100 μL effector cells + 50 μL culture medium), effector cell + antibody group (100 μL effector cells + 50 μL antibody + 50 μL culture medium), and blank control group (200 μL culture medium). The effector-to-target ratio was 10:1, and the cells were incubated for 24 hours. Centrifuge and collect the supernatant. Use the Human IL-2 Precoated ELISA Kit (Dayou, catalog number 1110203), Human IL-6 Precoated ELISA Kit (Dayou, catalog number 1110603), Human TNF-α Precoated ELISA Kit (Dayou, catalog number 1117203), and Human IFN-γ Precoated ELISA Kit (Dayou, catalog number 1110003) to detect and analyze the samples according to the kit instructions. Use an Infinite F50 microplate reader (model TECAN) for detection and analysis.

[0365] Figures 14A-14D Table 9 shows GC35-induced cytokine release in the presence of NCI-H929 cells.

[0366] Table 9. EC50 of cytokine release50 and maximum release

[0367] Example 16: Growth inhibition of NCI-H929 by anti-GPRC5D / anti-CD3 bispecific antibody in a mouse tumor model system The tumor-suppressive effect of GC35 in the NCI-H929 human multiple myeloma mouse xenograft model was investigated. NCI-H929 human myeloma cells were mixed with human PBMCs, and the resulting human PBMCs (0.5 × 10⁻⁶) were suspended in the mixture. 7 (cells / mL) and NCI-H929 cells (7.5 × 10⁻⁶) 7 A suspension of tumor cells (0.1 mL / mouse) was prepared and inoculated under aseptic conditions into the right axilla of B-NDG mice (source: Biocytogen). The diameter of the xenograft was measured with calipers, and the tumor was allowed to grow to 200 mm. 3 Animals were randomly divided into 7 groups of 6 mice each, with the grouping day designated as D0. The treatment group received the drug via tail vein injection (iv) at a volume of 10 mL / kg, while the control group received an equal volume of PBS solution. Drug administration was performed on D1, D4, D7, and D10, for a total of 4 administrations. The antitumor effect of the antibody was dynamically observed using tumor diameter measurement. Tumor volume was measured 2-3 times per week, and mouse weight was recorded. General behavior of the mice was observed and recorded daily.

[0368] Calculate each indicator using the following formula: Tumor volume TV (mm) 3 ) = 1 / 2 × (a × b 2 ), where a represents the long diameter of the tumor and b represents the short diameter of the tumor; Relative tumor volume RTV = TV t / TV0, where TV0 is the tumor volume on day D0, TV t The tumor volume at each measurement; Relative tumor proliferation rate T / C (%) = T RTV / C RTV × 100%, where T RTV For the treatment group RTV, C RTV RTV was used as the control group. Tumor inhibition rate (TGI) (%) = (1 - tumor weight in the treatment group / tumor weight in the control group) × 100%.

[0369] Experimental results are as follows Figure 15 As shown in Table 10, at high doses, Talquetamab and GC35 can significantly inhibit the growth of subcutaneous xenografts of human multiple myeloma NCI-H929, leading to tumor regression; at medium and low doses, GC35 is more effective than Talquetamab.

[0370] Table 10. Effects of anti-GPRC5D / anti-CD3 bispecific antibody on subcutaneous xenograft tumors of NCI-H929 human myeloma cells in NDG mice

[0371] If the tumor volume is smaller than that at the time of grouping, i.e., TV t < TV0, it is considered "tumor regression"; "tumor disappearance" means complete disappearance of the tumor, and at this time, TGI (%) = 100%.

[0372] The sequence information of the present disclosure is summarized in Table S2 below.

[0373]

[0374] For the purposes of description and disclosure, all patents, patent applications, and other identified publications are hereby expressly incorporated herein by reference. These publications are provided only because their disclosure predates the filing date of the present disclosure. All statements as to the dates of these documents or the representation of the content of these documents are based on the information available to the applicant and do not constitute any admission as to the dates of these documents or the correctness of the content of these documents. Moreover, in any country, any reference to these publications in this text does not constitute an admission that such publication is part of the common general knowledge in the art.

[0375] Although the present disclosure has been described in detail above by means of general descriptions and specific embodiments, modifications or improvements can be made thereto on the basis of the present disclosure, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present disclosure all fall within the scope of protection required by the present disclosure.

Claims

1. A bispecific antibody comprising a first antigen-binding moiety binding to GPRC5D and a second antigen-binding moiety binding to CD3, wherein, The first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, 9, 17, 25 or 33; HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, 59, 10, 18, 26 or 34; HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, 11, 19, 27 or 35; LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, 12, 20, 28 or 36; LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, 13, 21, 29 or 37; and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6, 14, 22, 30 or 38.

2. The bispecific antibody according to claim 1, wherein, The first antigen-binding portion comprises: (1) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; wherein X1 is selected from D or E, and X2 is selected from G or A; (2) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (3) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; HCDR2 containing the amino acid sequence shown in SEQ ID NO: 3, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (4) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 61, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (5) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 9, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 10, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 11, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

6. LCDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 14;(6) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 17, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 18, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 22; (7) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 27, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30; or (8) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

17. HCDR3, containing the amino acid sequence shown in SEQ ID NO: 36, LCDR1, containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3, containing the amino acid sequence shown in SEQ ID NO:

38.

3. A bispecific antibody comprising a first antigen-binding moiety binding to GPRC5D and a second antigen-binding moiety binding to CD3, wherein, The first antigen-binding region comprises: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, 15, 23, 31, 39, 66 or 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8, 16, 24, 32, 40, 67 or 69.

4. The bispecific antibody according to claim 3, wherein, The first antigen-binding region comprises: (1) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 8; (2) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 16; (3) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 24; (4) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:

31. (5) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 39, and HCDR1, HCDR2, and HCDR3 in the light chain variable region as shown in SEQ ID NO: 40; (6) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 62, and HCDR1, HCDR2, and HCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (7) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 63, and HCDR1, HCDR2, and HCDR3 in the light chain variable region as shown in SEQ ID NO: 67; (8) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 64, and HCDR1, HCDR2, and HCDR3 in the light chain variable region as shown in SEQ ID NO:

64. (9) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 65, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67.(10) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 66, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 67, wherein X3 is selected from Q or V, X4 is selected from P or A, X5 is selected from L or V, X6 is selected from K or A, X7 is selected from I or M, X8 is selected from D or E, X9 is selected from G or A, X; 10 Selected from K or R, X 11 Selected from A or V, X 12 Selected from K or T, X 13 Selected from S or A, X 14 Selected from T or R, X 15 Selected from S or T, X 16 Selected from A or Q; or (11) HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 68, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

69.

5. The bispecific antibody according to any one of claims 1-4, wherein, The first antigen-binding portion comprises a heavy chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 7, 15, 23, 31, 39, 62, 63, 64, 95%, 97%, 98%, 99%, or 100%; and a light chain variable region having an amino acid sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to the amino acid sequence shown in SEQ ID NO: 8, 16, 24, 32, 40, 67, or 69.

6. The bispecific antibody according to any one of claims 1-5, wherein, The first antigen-binding region comprises: (1) a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 8; (2) a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 15, and a light ... The amino acid sequence shown in SEQ ID NO: 16 has a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 23; (3) a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 24; and a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 24; (4) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 24; The amino acid sequence shown in SEQ ID NO: 31 has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 32;(5) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 40; (6) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 62, and a light ... The amino acid sequence shown in SEQ ID NO: 67 has a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 63; (7) a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; and a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; (8) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67; The amino acid sequence shown in SEQ ID NO: 64 has a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 67;(9) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; (10) A heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 66, and a light ...7, and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, The amino acid sequence shown in SEQ ID NO: 67 has a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 68; or (11) a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 69; and a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:

69.

7. The bispecific antibody according to any one of claims 1-6, wherein, The first antigen-binding region comprises: (1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; (2) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16; (3) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24; (4) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 32; (5) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40; (6) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67; (7) a region comprising the amino acid sequence shown in SEQ ID NO: 68, and a region comprising the amino acid sequence shown in SEQ ID NO: 69, and a region comprising the amino acid sequence shown in SEQ ID NO: 60 ... (6) The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 63, and the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (8) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 64, and the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (9) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 65, and the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (10) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 66, and the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; or (11) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68, and the light chain variable region containing the amino acid sequence shown in SEQ ID NO:

69.

8. The bispecific antibody according to claim 1, wherein, The first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 1, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 2, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 3, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 4, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

6.

9. The bispecific antibody according to claim 8, wherein, The first antigen-binding portion comprises: a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 62; and a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 67; preferably, the first antigen-binding portion comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 62; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

67.

10. The bispecific antibody according to claim 1, wherein, The first antigen-binding portion comprises: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34, HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR1 containing the amino acid sequence shown in SEQ ID NO: 36, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38.

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