Bispecific antibodies targeting G protein-coupled receptors

Abstract

A bispecific antibody targeting a G protein-coupled receptor is provided. Specifically, a pharmaceutical composition comprising a bispecific antibody, an encoding nucleic acid, a vector containing the nucleic acid, cells containing the vector, and the bispecific antibody is provided, as well as their use in the treatment of related diseases in subjects.

Description

[Technical Field]

[0001] cross reference This application claims the rights of Chinese Patent Application No. CN202310484445.0, filed on 28 April 2023, the contents of which are incorporated herein by reference in whole.

[0002] Sequence listing file submitted simultaneously The entire contents of the following XML file are incorporated herein by reference: Sequence listing in computer-readable format (CRF) (name: TFH01011PCT-sequence listing.xml, date: 20240424, size: 119KB).

[0003] This disclosure relates to bispecific antibodies, particularly bispecific antibodies targeting GPRC5D and CD3. This disclosure also relates to methods for preparing and using such bispecific antibodies. [Background technology]

[0004] Multiple myeloma (MM) is the second most common hematological malignancy after non-Hodgkin lymphoma. Its pathological features include the abnormal proliferation of plasma cells in the bone marrow, which inhibits the development of normal blood cells, leading to cytopenia, bone damage, and kidney damage. In 2016, an estimated 16,500 new cases and 10,300 deaths of multiple myeloma were reported in China, with an estimated incidence of 1.6 cases per 100,000 people.

[0005] GPRC5D (G protein-coupled receptor class C group 5 member D) is a 7-transmembrane protein composed of 345 amino acid residues, belonging to the retinoic acid-inducible orphan G protein-coupled receptor (RAIG) family. While its normal physiological function is related to the structure of hard keratin, the specific biological function and ligands of GPRC5D remain unclear. Several studies have shown that the expression threshold for GPRC5D exceeds 50% in malignant myeloid plasma cells of 65% of multiple myeloma patients, and that 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. GPRC5D is either not expressed at all or at very low levels in normal tissues, except in hair follicle tissue of the skin and myeloid plasma cells, highlighting its potential clinical value as it could offer a new therapeutic option for multiple myeloma patients.

[0006] While several antibodies targeting GPRC5D are already under development as a potential therapeutic target, their options are still limited, and more choices are needed.

[0007] This disclosure provides bispecific antibodies targeting GPRC5D and CD3, as well as related nucleic acids, vectors, cells, pharmaceutical compositions, preparation methods, and uses that can encode the provided antibodies.

[0008] In one embodiment, the present disclosure provides a bispecific antibody comprising a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety is (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 Includes.

[0009] In one embodiment, the Disclosure also provides a bispecific antibody comprising a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety is (1) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8; (2) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16; (3) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24; (4) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 32; (5) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 39, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40; (6) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (7) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (8) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (9) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 65, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (10) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; or (11) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 69 Includes.

[0010] In some embodiments, the antigen-binding portion of the first embodiment is (1) A heavy chain variable region whose amino acid sequence 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; and a light chain variable region whose amino acid sequence 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 whose amino acid sequence 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 whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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) A heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 whose amino acid sequence 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; and a light chain variable region whose amino acid sequence 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; (6) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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; (7) Heavy chain variable regions whose amino acid sequence 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, and light chain variable regions whose amino acid sequence 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) A heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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; (10) A heavy chain variable region whose amino acid sequence 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: 66, and a light chain variable region whose amino acid sequence 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; or (11) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 Includes.

[0011] In some embodiments, the second antigen-binding portion includes 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.

[0012] In some embodiments, the second antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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.

[0013] In some specific embodiments, the bispecific antibody comprises a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety 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, and the second antigen-binding moiety 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 moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety 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, and the second antigen-binding moiety 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.

[0015] In some specific embodiments, the bispecific antibody comprises a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67, and the second antigen-binding moiety 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.

[0016] In other specific embodiments, the bispecific antibody comprises a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69, and the second antigen-binding moiety 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.

[0017] In some specific embodiments, the bispecific antibodies of this disclosure include an Fc domain comprising two Fc polypeptides.

[0018] In some specific embodiments, the first antigen-binding portion and the second antigen-binding portion are, independently, Fab, scFv, scFab (single-stranded Fab), or other structures. In some embodiments, the first antigen-binding portion is Fab, and the second antigen-binding portion is scFv.

[0019] In some embodiments, the bispecific antibody of the present disclosure has a first antigen-binding moiety of Fab and a second antigen-binding moiety of scFv, wherein the C-terminus of the Fab heavy chain or Fab light chain of the first antigen-binding moiety is fused to the N-terminus of one Fc polypeptide of the Fc domain, and the C-terminus of the second antigen-binding moiety is fused to the N-terminus of the other Fc polypeptide of the Fc domain. In a more specific embodiment, the bispecific antibody is composed of three polypeptide chains, the first polypeptide chain comprising the Fab light chain of the first antigen-binding moiety, the second polypeptide chain comprising the Fab heavy chain of the first antigen-binding moiety and one Fc polypeptide of the Fc domain, and the third polypeptide chain comprising the second antigen-binding moiety and the other Fc polypeptide of the Fc domain.

[0020] In some embodiments, the bispecific antibody is bivalent.

[0021] In another aspect, this disclosure is: (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 The present invention provides an antibody or its antigen-binding moiety that binds to GPRC5D, including the antibody itself.

[0022] In one aspect, this disclosure also, (1) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8; (2) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16; (3) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24; (4) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 32; (5) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 39, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40; (6) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (7) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (8) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (9) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 65, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (10) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; or (11) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 69 The present invention provides an antibody or its antigen-binding moiety that binds to GPRC5D, including the antibody itself.

[0023] In some embodiments, the antibody or antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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, 63, 64, 65, 66, or 68, and a light chain variable region whose amino acid sequence 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 or 69.

[0024] In some embodiments, the antibody or its antigen-binding portion that binds to GPRC5D is (1) A heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 whose amino acid sequence 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, and a light chain variable region whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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; (4) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 whose amino acid sequence 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: 66, and a light chain variable region whose amino acid sequence 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; or (6) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 Includes.

[0025] In some embodiments, the antibody or its antigen-binding portion that binds to GPRC5D is (1) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (2) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (3) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (4) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (5) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68, and light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69 Includes.

[0026] In one embodiment, the Disclosure provides an isolated nucleic acid comprising a bispecific antibody or a nucleotide sequence encoding an antibody or its antigen-binding portion as described herein.

[0027] In one embodiment, the Disclosure provides a vector comprising the nucleic acid described herein.

[0028] In one embodiment, the Disclosure provides a host cell comprising the nucleic acid or vector described herein.

[0029] In one embodiment, the present disclosure provides an antibody-drug conjugate comprising an antibody or its antigen-binding moiety that binds to GPRC5D as described in the present disclosure.

[0030] In one embodiment, the Disclosure provides an anti-GPRC5D chimeric antigen receptor comprising an antibody that binds to GPRC5D as described herein.

[0031] In one embodiment, the present disclosure provides engineered immune effector cells comprising the chimeric antigen receptor described herein.

[0032] In another aspect, the Disclosure provides a method for preparing a bispecific antibody or an antibody or its antigen-binding moiety as described herein, the method comprising culturing host cells to express a bispecific antibody or an antibody or its antigen-binding moiety, and isolating and purifying the bispecific antibody or an antibody or its antigen-binding moiety in system.

[0033] In another embodiment, the present disclosure provides a pharmaceutical composition comprising the antibody or its antigen-binding moiety, the antibody-drug conjugate, the engineered immunoeffector cells, or the bispecific antibody, and a pharmaceutically acceptable additive.

[0034] In another aspect, the disclosure provides the use of the antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immunoeffector cells, the bispecific antibody, or the pharmaceutical composition in the preparation of a drug for treating a disease expressing GPRC5D.

[0035] In another aspect, the Disclosure provides a method for treating a disease expressing GPRC5D, the method comprising administering the antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immune effector cells, the bispecific antibody, or the pharmaceutical composition to a subject in need. In another aspect, the Disclosure provides a method for treating a disease expressing GPRC5D, comprising administering the therapeutically effective amount of the antibody or its antigen-binding portion, the antibody-drug conjugate, the engineered immune effector cells, the bispecific antibody, or the pharmaceutical composition to a subject in need.

[0036] The bispecific antibodies targeting GPRC5D and CD3 provided in this disclosure exhibit favorable antitumor efficacy and / or safety. [Brief explanation of the drawing]

[0037] [Figure 1]This figure shows the OD450nm of the binding of certain mouse anti-hGPRC5D antibodies to CHO-K1-hGPRC5Dhi cells, CHO-K1-cynoGPRC5D cells, and CHO-K1-murineGPRC5D cells, as measured by ELISA. [Figure 2] This figure shows the MFI and EC50 values ​​for FACS detection of chimeric antibodies that bind to cells with high hGPRC5D expression. [Figure 3] This figure shows the MFI (Multiple First Inspection) of FACS detection of a chimeric antibody that binds to cells with low hGPRC5D expression. [Figure 4A] This figure shows the MFI detection of anti-GPRC5D antibodies that bind to different cells using FACS, with A being a figure of MFI bound to NCI-H929 cells. [Figure 4B] This figure shows the FACS detection of MFI of anti-GPRC5D antibodies that bind to different cells, with B showing MFI bound to MM.1S cells. [Figure 5A] This figure shows the multi-cell infiltration (MFI) of FACS detection of anti-GPRC5D antibodies that bind to different cell types, where A represents CHO-K1-cynoGPRC5D cells. [Figure 5B] This figure shows the multi-cell infiltration (MFI) of FACS detection of anti-GPRC5D antibodies that bind to different cell types, where B represents CHO-K1-murineGPRC5D cells. [Figure 6A] This figure shows the multi-cell infiltration (MFI) of FACS detection of anti-GPRC5D antibodies that bind to different cell types, where A represents CHO-K1-GPRC5A cells. [Figure 6B] This figure shows the multi-cell infiltration (MFI) of FACS detection of anti-GPRC5D antibodies that bind to different cell types, where B represents CHO-K1-GPRC5B cells. [Figure 6C] This figure shows the multi-cell infiltration (MFI) of FACS detection of anti-GPRC5D antibodies that bind to different cell types, where C represents CHO-K1-GPRC5C cells. [Figure 7] This figure shows the MFI (Multiple First Injection) of FACS detection of anti-GPRC5D antibody binding to CHO-K1 cells. [Figure 8]This figure shows the MFI (Multiple Filtration Indication) of chimeric and humanized anti-GPRC5D antibodies to NCI-H929 cells as measured by FACS. [Figure 9] This figure shows the structure of an exemplary bispecific antibody in this disclosure. [Figure 10A] This figure shows the multi-cell infiltration (MFI) of FACS detection of anti-GPRC5D / anti-CD3 bispecific antibodies that bind to different cell types, where A represents RPMI-8226 cells. [Figure 10B] This figure shows the MFI (Multiple Cell Injection Finding) of FACS detection of anti-GPRC5D / anti-CD3 bispecific antibodies that bind to different cell types, where B represents MM.1S cells. [Figure 10C] This figure shows the multi-cell infiltration (MFI) of FACS detection of anti-GPRC5D / anti-CD3 bispecific antibodies that bind to different cell types, where C represents NCI-H929 cells. [Figure 11] This figure shows the MFI (Multiple Filtration Indication) of GPRC5D / anti-CD3 bispecific antibody binding to CD3+ T cells as measured by FACS. [Figure 12A] This figure shows the lysis rates of anti-GPRC5D / anti-CD3 bispecific antibodies against different cell types, with A being NCI-H929 cells. [Figure 12B] This figure shows the lysis rates of anti-GPRC5D / anti-CD3 bispecific antibodies against different cell types, where B represents MM.1S cells. [Figure 12C] This figure shows the lysis rates of anti-GPRC5D / anti-CD3 bispecific antibodies against different cell types, where C represents RPMI-8226 cells. [Figure 13A] This figure shows the effect of anti-GPRC5D / anti-CD3 bispecific antibody on T cell activation in the presence of target cell MM.1S, where CD69 and CD25 are T cell activation markers. A is the ratio of CD25+ cells to CD4+ cells. [Figure 13B] This figure shows the effect of anti-GPRC5D / anti-CD3 bispecific antibody on T cell activation in the presence of target cell MM.1S, where CD69 and CD25 are T cell activation markers. B is the ratio of CD69+ cells to CD4+ cells. [Figure 13C]This figure shows the effect of anti-GPRC5D / anti-CD3 bispecific antibody on T cell activation in the presence of target cell MM.1S, where CD69 and CD25 are T cell activation markers. C is the ratio of CD25+ cells to CD8+ cells. [Figure 13D] This figure shows the effect of anti-GPRC5D / anti-CD3 bispecific antibody on T cell activation in the presence of target cell MM.1S, where CD69 and CD25 are T cell activation markers. D is the ratio of CD69+ cells to CD8+ cells. [Figure 14A] This figure shows the release levels of different cytokines after co-incubating target cells MM.1S and effector cells PBMC with different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody for 24 hours. A is IL-2. [Figure 14B] This figure shows the release levels of different cytokines after co-incubating target cells MM.1S and effector cells PBMC with different concentrations of anti-GPRC5D / anti-CD3 bispecific antibodies for 24 hours. B is IL-6. [Figure 14C] This figure shows the release levels of different cytokines after co-incubating target cells MM.1S and effector cells PBMC with different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody for 24 hours. C is TNF-α. [Figure 14D] This figure shows the release levels of different cytokines after co-incubating target cells MM.1S and effector cells PBMC with different concentrations of anti-GPRC5D / anti-CD3 bispecific antibody for 24 hours. D is IFN-γ. [Figure 15] This figure shows the effect of anti-GPRC5D / anti-CD3 bispecific antibodies on tumor volume in a mouse xenograft model of NCI-H929 human multiple myeloma. [Modes for carrying out the invention]

[0038] I. Definitions and Explanations Unless otherwise specified, the following terms used in this disclosure have the meanings set forth below. Where a particular term is not given a specific definition, it should not be construed as ambiguous or unclear, but rather should be understood in accordance with its common meaning in the art. Where a trade name is mentioned herein, it refers to the corresponding product or its active ingredient.

[0039] The term "antibody," used in its broadest sense, covers natural and artificial antibodies of various structures and encompasses multiple antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), and single-chain antibodies, as long as they exhibit the desired antigen-binding activity.

[0040] The term "multispecificity" refers to an antibody's ability to specifically bind to multiple different antigenic determinants, such as two or more (i.e., "bispecific") antigenic determinants. In this specification, antigenic determinant is synonymous with antigenic epitope. Generally, a bispecific antibody has two antigen-binding sites, each specific to a different antigenic determinant. These different antigenic determinants can be expressed on the same or different cells. The different antigenic determinants may differ depending on the type of antigen (e.g., binding to antigens GPRC5D and CD3), or they may be present on the same antigen. An antigenic determinant is a specific chemical group with a particular composition and structure located on or elsewhere on the surface of an antigenic substance molecule, capable of specifically binding to the corresponding antibody or sensitized lymphocyte. An example of an antigenic determinant is GPRC5D, which, as an antigenic substance, has multiple structurally defined or undefined antigenic determinants. A particular bispecific antibody can, for example, bind to both GPRC5D and CD3.

[0041] The term "antigen-binding site" refers to a polypeptide molecule that specifically binds to an antigenic determinant. Specific examples of antigen-binding sites include Fab, scFv, or scFab.

[0042] In this disclosure, the terms “first,” “second,” and “third” used with respect to antigen-binding sites, antigens, Fc polypeptides, peptide linkers, polypeptide chains, etc., are used for convenience to distinguish cases where one or more parts of each type are present. Unless expressly stated otherwise, the use of these terms is not intended to confer any particular order or orientation.

[0043] The term "fusion" refers to the linking of constituent elements (e.g., antigen-binding moieties, Fc polypeptides, etc.) by peptide bonds, either directly or via one or more peptide linkers. For example, some peptide linkers consist of 1 to 50 amino acids linked by peptide bonds, and these amino acids can be selected from 20 native amino acids. In a more preferred embodiment, the 1 to 50 amino acids are selected from glycine, alanine, proline, serine, asparagine, glutamine, and lysine.

[0044] The term "variable domain" or "variable region" refers to an antibody domain involved in the binding of an antibody to an antigen. For example, natural four-chain antibodies (e.g., derived from humans, mice, etc.) have a heavy chain variable region (also called the heavy chain variable domain, VH, or VH domain) and a light chain variable region (also called the light chain variable domain, VL, or VL domain). In most cases, each variable domain of a natural antibody is essentially composed of four "framework regions (FRs)" and three "complementarity-determining regions (CDRs)". The four framework regions are called framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4), respectively, and these framework regions are separated by three complementarity-determining regions, which are called complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3), respectively, in the Art and below. Therefore, the general structure of the variable domain can be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain confers antigen specificity to the antibody by containing an antigen-binding site.

[0045] The term "complementarity-determining region" (CDR) is also called "hypervariable region" (HVR). For example, a natural quadruple-chain antibody typically contains six CDRs, three of which are located in the heavy chain variable region: heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and the remaining three are located in the light chain variable region: light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3).

[0046] Currently, there are numerous methods for classifying and defining CDRs. Among them, the Kabat definition, which classifies CDRs based on sequence diversity, is the most commonly used (Elvin A. Kabat, et al, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Chothia definition is based on the position 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 Oxford Molecular's AbM antibody modeling software. The "contact" CDR definition is based on the analysis of the crystal structure of available complexes. There is also a CCG definition. However, it should be noted that the boundaries of the CDRs of the same antibody variable region defined based on different methods may differ, meaning that the CDR sequences of the same antibody variable region defined by different methods may differ. Therefore, when limiting an antibody using a specific CDR sequence, the range of the antibody may also include antibodies defined by CDR sequences of any other arbitrary definition (e.g., one or more combinations of Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0047] 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 specification, Fab refers to a native or modified Fab molecule, that is, a Fab heavy chain consisting of a variable region VH and a constant region CH1 (VH-CH1, from N-terminus to C-terminus) and a Fab light chain consisting of a variable region VL and a constant region CL (VL-CL, from N-terminus to C-terminus). A modified Fab may, for example, have amino acid substitutions introduced into the CH1 / CL domain and / or the VH / VL domain. Specifically, a modified Fab may have amino acid substitutions introduced into the CL domain.

[0048] The term "scFv" encompasses the VH and VL domains of an immunoglobulin, and these domains are present within a single polypeptide chain. In some embodiments, the scFv further includes a peptide linker between the VH and VL domains, thereby allowing the scFv to form a desired structure for antigen binding. In a specific example, the scFv is arranged from the N-terminus to the C-terminus in the order of VL-linker-VH.

[0049] The terms “Fc structural region,” “Fc,” or “Fc domain” are used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term encompasses both the native sequence Fc and mutant Fc. The C-terminal lysine (Lys447) of Fc may or may not be present. Unless otherwise noted, the numbering of amino acid residues in Fc or the constant region follows the EU numbering system (also known as the EU index) described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. In this specification, “Fc polypeptide” of one of the Fc domains refers to one of the two polypeptides that form a dimeric Fc domain. For example, the Fc polypeptide of the IgG Fc domain includes IgG CH2 and IgG CH3.

[0050] The term "treatment" means administering any compound (e.g., monospecific antibody, bispecific antibody) or pharmaceutical composition described herein to prevent, improve, or eliminate one or more symptoms of a disease or associated with such disease, including, but not limited to, (i) preventing the development of a disease or condition in a mammal (especially if the mammal is susceptible to the disease or condition but has not yet been diagnosed with it), (ii) inhibiting the development of a disease or condition, (iii) alleviating a disease or condition, i.e., causing regression of the disease or condition, and (iv) mitigating the direct or indirect pathological effects of a disease or condition.

[0051] The term “therapeutic dose” means (i) a dose that treats or prevents a particular disease, condition, or disorder; (ii) a dose that alleviates, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder; or (iii) a dose that prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. The amount constituting the “therapeutic dose” of a monospecific antibody, bispecific antibody, antibody-drug conjugate, engineered immune effector cell, or pharmaceutical composition of this disclosure may vary depending on factors such as the compound or pharmaceutical composition and its ability to induce 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 being treated. The therapeutic dose may also be routinely determined by a person skilled in the art based on their knowledge and this disclosure.

[0052] The term "pharmaceutically acceptable" means that, within the bounds of sound medical judgment, a compound, material, composition, and / or dosage form is suitable for use in contact with human and animal tissues in a reasonable benefit-to-risk ratio without causing excessive toxicity, irritation, allergic reactions, or other problems or complications.

[0053] The term "excipient" refers to components other than the active ingredient (e.g., the antibody in this disclosure). The selection of an excipient largely depends on factors such as the specific route of administration, the effectiveness of the excipient in determining solubility and stability, and the properties of the dosage form.

[0054] The term "isolated" refers to a target compound that has been separated from its natural environment, such as an antibody or its antigen-binding fragment, or a nucleic acid.

[0055] The terms "Xn" and "Xaa" are equivalent and refer to an unspecified amino acid, the range of which is specified by the subsequent definition in the relevant expression.

[0056] When used herein, "EC 50", that is, the maximum half-value effective concentration, refers to the concentration of the antibody that induces a response that is 50% of the maximum response, that is, the response midway between the maximum response and the baseline. EC 50 can be measured by ELISA or FACS analysis, or any other method known in the art.

[0057] "K D " refers to the equilibrium dissociation constant, which is the ratio of the dissociation rate constant (k d ) to the association rate constant (k a ), that is, k d / k a ), and is expressed in molar concentration (M). The K D value of an antibody can be measured using methods established in the art. A preferred method for measuring the K D of an antibody is to use surface plasmon resonance (SPR) technology, preferably a biosensor system such as a Biacore surface plasmon resonance system.

[0058] The term "identity" is also referred to as consistency. The "percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues that are identical to the amino acid residues of a specific amino acid sequence shown herein when the aligned sequences are aligned with the specific amino acid sequence shown herein, and gaps are introduced as necessary to achieve the maximum percent sequence identity, and no conservative substitutions are made. The identity alignment of amino acid sequences can be performed using various methods within the scope of the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to obtain the maximum alignment over the full length of the sequences being compared.

[0059] 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, for example, non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. Preferably, the subjects as described herein are human. Unless otherwise specified, the terms “patient” and “subject” are used interchangeably. “Subjects who require it” include subjects who already have a disease or condition, subjects at risk of developing a disease or condition, and subjects at risk of developing a disease or condition for which prevention, delay, or reduction of the disease or condition is intended.

[0060] In this specification, “approximately” means within the tolerance range for a particular value as determined by those skilled in the art, which depends in part on the limits of the method of measuring or determining the value, i.e., the measuring system. For example, “approximately” may mean within a standard deviation of 1 or more, as is practice in the art. Alternatively, “approximately” may mean within a range of up to ±5% of a given particular value, e.g., ±2%, ±1%, or ±0.5%. Where a particular value is given within the scope of this disclosure, unless otherwise specified, “approximately” should be interpreted as meaning within the tolerance range for that particular value. Unless otherwise specified in this specification, step parameter or condition values ​​are qualified with “approximately” by default.

[0061] The terms “comprise,” “comprises,” or “comprising,” and their synonyms (e.g., contain, contains, containing, include, includes, and including), should be understood as “including but not limited to,” meaning to cover other unspecified elements, components, and steps in addition to those listed.

[0062] In this specification, unless explicitly indicated otherwise in the context, singular terms encompass plural referents, and vice versa.

[0063] II. Single-specific antibodies or their antigen-binding moieties This disclosure provides a monospecific antibody or its antigen-binding moiety that binds to GPRC5D, comprising HCDR1 containing the amino acid sequence shown in SEQ ID NOs. 1, 9, 17, 25, or 33; HCDR2 containing the amino acid sequence shown in SEQ ID NOs. 59, 10, 18, 26, or 34; HCDR3 containing the amino acid sequence shown in SEQ ID NOs. 3, 11, 19, 27, or 35; LCDR1 containing the amino acid sequence shown in SEQ ID NOs. 4, 12, 20, 28, or 36; LCDR2 containing the amino acid sequence shown in SEQ ID NOs. 5, 13, 21, 29, or 37; and LCDR3 containing the amino acid sequence shown in SEQ ID NOs. 6, 14, 22, 30, or 38.

[0064] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes 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, where 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.

[0065] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes 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.

[0066] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes 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.

[0067] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes 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.

[0068] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes 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.

[0069] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes 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.

[0070] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes 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.

[0071] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes 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.

[0072] The disclosure also provides a monospecific antibody or its antigen-binding moiety that binds to GPRC5D, comprising HCDR1, HCDR2, and HCDR3 in a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NOs. 7, 15, 23, 31, 39, 66, or 68, and LCDR1, LCDR2, and LCDR3 in a light chain variable region comprising the amino acid sequence shown in SEQ ID NOs. 8, 16, 24, 32, 40, 67, or 69.

[0073] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0074] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16.

[0075] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24.

[0076] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 32.

[0077] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 39, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40.

[0078] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0079] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0080] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0081] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 65, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0082] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67, where 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 is selected from K or R, X 11 is selected from A or V, X 12 is selected from K or T, X 13 It is selected from S or A, X 14 is selected from T or R, X 15 It is selected from S or T, X 16The answer is selected from A or Q.

[0083] In some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 69.

[0084] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 NOs. 7, 15, 23, 31, 39, 62, 63, 64, 65, 66, or 68, and a light chain variable region whose amino acid sequence 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 NOs. 8, 16, 24, 32, 40, 67, or 69.

[0085] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0086] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0087] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 monospecific antibody or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0088] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 monospecific antibody or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0089] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 monospecific antibody or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0090] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 monospecific antibody or its antigen-binding moiety that binds to GPRC5D further includes 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 or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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, and a light chain variable region whose amino acid sequence 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 monospecific antibody or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0092] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 monospecific antibody or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0093] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 monospecific antibody or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0094] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D has a heavy chain variable region whose amino acid sequence 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: 66, and an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 67. , includes a light chain variable region having identity of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, where 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 is selected from K or R, X 11 is selected from A or V, X 12 is selected from K or T, X 13It is selected from S or A, X 14 is selected from T or R, X 15 It is selected from S or T, X 16 X1 is selected from A or Q. Furthermore, in some such embodiments, the monospecific antibody or its antigen-binding moiety that binds to the GPRC5D 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: 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: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, where X1 is selected from D or E, and X2 is selected from G or A.

[0095] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 or its antigen-binding moiety that binds to GPRC5D further includes 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.

[0096] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 8.

[0097] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 16.

[0098] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 24.

[0099] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 31 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 32.

[0100] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 39 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 40.

[0101] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67.

[0102] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 63 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67.

[0103] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 64 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67.

[0104] In some embodiments, the single-specific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 65 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67.

[0105] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 66 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67.

[0106] In some embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69.

[0107] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0108] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16.

[0109] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24.

[0110] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 32.

[0111] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 39 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40.

[0112] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 62 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0113] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0114] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 64 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0115] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 65 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0116] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 66 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67.

[0117] In some specific embodiments, the monospecific antibody or its antigen-binding portion that binds to GPRC5D includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 68 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 69.

[0118] In this disclosure, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D is a mouse antibody, a chimeric antibody, or a humanized antibody. Humanization can reduce immunogenicity. Therefore, in some embodiments, the monospecific antibody or its antigen-binding moiety that binds to GPRC5D is humanized.

[0119] In some embodiments, the monospecific antibody that binds to GPRC5D of this disclosure may also include an immunoglobulin constant region, or a fragment, analog, variant, or derivative of the constant region. In some embodiments, the constant region includes a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is derived from a human immunoglobulin heavy chain such as IgG1, IgG2, IgG3, or IgG4, or another class of immunoglobulin heavy chain. In some embodiments, the light chain constant region is derived from a human immunoglobulin light chain such as a κ light chain or λ light chain of human immunoglobulin. In some embodiments, the constant region may include any modifications disclosed herein or known in the art, such as amino acid insertions, deletions, substitutions, or chemical modifications. In some embodiments, the constant region may include mutations that alter effector function. In some embodiments, any amino acid residue in the constant region may be substituted with any allotype amino acid residue. In some embodiments, the C-terminal lysine (Lys447) in the heavy chain constant region may or may not be present. In some specific embodiments, the monospecific antibody that binds to GPRC5D comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 70 and a light chain containing the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the monospecific antibody that binds to GPRC5D comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 72 and a light chain containing the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the monospecific antibody that binds to GPRC5D comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 74 and a light chain containing the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the monospecific antibody that binds to GPRC5D comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 76 and a light chain containing the amino acid sequence shown in SEQ ID NO: 78. In some specific embodiments, the monospecific antibody that binds to GPRC5D comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 80 and a light chain containing the amino acid sequence shown in SEQ ID NO: 82. In some embodiments, the C-terminal lysine of the heavy chain is absent.

[0120] Tables S1 and S2 provide CDR and variable region amino acid sequence information for several exemplary monospecific antibodies that bind to GPRC5D (e.g., chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, and Chi-169, as well as 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. [Table 1]

[0121] The monospecific antibody or its antigen-binding moiety that binds to GPRC5D according to this disclosure 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 to GPRC5A, GPRC5B, and GPRC5C, or substantially does not bind to them.

[0122] Isolated nucleic acids This disclosure provides isolated nucleic acids comprising a monospecific antibody that binds to GPRC5D as described herein, or a nucleotide sequence encoding the antigen-binding portion thereof. The sequence listing exemplifies several GPRC5D-binding monospecific antibodies or nucleotide sequences encoding the antigen-binding portions thereof.

[0123] vector This disclosure provides a vector comprising nucleic acid. In some embodiments, the vector is a cloning vector, and in other embodiments, the vector is an expression vector, specifically, the expression vector being pcDNA3.1(+). The expression vector may optionally be any expression vector capable of expressing a monospecific antibody that binds to GPRC5D as described in this disclosure.

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

[0125] Antibody-drug conjugate (ADC) This disclosure provides an antibody-drug conjugate comprising a monospecific antibody or its antigen-binding moiety that binds to GPRC5D of this disclosure. In some embodiments, the antibody-drug conjugate comprises a monospecific antibody or its antigen-binding moiety that binds to GPRC5D and a cytotoxic agent. The monospecific antibody or its antigen-binding moiety that binds to GPRC5D and the cytotoxic agent are preferably linked by a linker, which may be a cleavable or incleavable linker.

[0126] Chimeric antigen receptor (CAR) The disclosure also provides an anti-GPRC5D chimeric antigen receptor (CAR) comprising a monospecific antibody or its antigen-binding moiety that binds to GPRC5D as disclosed herein.

[0127] Anti-GPRC5D CARs may include (a) an extracellular antigen-binding domain that binds to GPRC5D, (b) a transmembrane domain, and (c) an intracellular signaling domain.

[0128] This disclosure also provides engineered immune effector cells, including CARs 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 a pharmaceutical composition comprising a monospecific antibody or its antigen-binding portion, an antibody-drug conjugate, or engineered immunoeffector cells that bind to GPRC5D of this disclosure, and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulants, fillers, buffers, or other agents.

[0130] Method for preparing a monospecific antibody or its antigen-binding moiety. In some embodiments, the Disclosure provides a method for preparing a monospecific antibody or its antigen-binding moiety that binds to GPRC5D, the method comprising culturing host cells to express the monospecific antibody or its antigen-binding moiety, and isolating and purifying the monospecific antibody or its antigen-binding moiety in system. To produce the monospecific antibody or its antigen-binding moiety, a nucleic acid encoding the monospecific antibody or its antigen-binding moiety is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be obtained using various methods known in the Art, such as gene splicing and chemical synthesis.

[0131] The purity of a single-specific antibody or its antigen-binding moiety can be determined by any of the following well-known analytical methods: gel electrophoresis, high-performance liquid chromatography, size exclusion chromatography, etc.

[0132] The physicochemical properties and / or biological activity of the monospecific antibodies or their antigen-binding moieties in this disclosure can be identified, screened, or characterized by a variety of assays 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, and / or hu128-v1.

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

[0135] This disclosure provides a method for treating a disease expressing GPRC5D, the method comprising administering the monospecific antibody or its antigen-binding moiety, the antibody-drug conjugate, the engineered immune effector cells, or the pharmaceutical composition to a subject in need. This disclosure provides a method for treating a disease expressing GPRC5D, comprising administering a therapeutically effective amount of the monospecific antibody or its antigen-binding moiety, the antibody-drug conjugate, the engineered immune effector cells, or the pharmaceutical composition to a subject in need. 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 hematological malignancy. In some embodiments, the tumor is a B-cell lymphoma. In some embodiments, the tumor is a B-cell lymphoma expressing GPRC5D. In some embodiments, the tumor is multiple myeloma (MM). In some embodiments, the tumor is a multiple myeloma expressing GPRC5D.

[0137] In some embodiments, 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 moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety includes 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 moiety includes 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, where 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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] The disclosure also provides a bispecific antibody comprising a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NOs. 7, 15, 23, 31, 39, 66, or 68, and LCDR1, LCDR2, and LCDR3 in a light chain variable region consisting of the amino acid sequence shown in SEQ ID NOs. 8, 16, 24, 32, 40, 67, or 69.

[0148] In some embodiments, the first antigen-binding region includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0149] In some embodiments, the first antigen-binding region includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16.

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

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

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

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

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

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

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

[0157] In some embodiments, the first antigen-binding region comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67, where 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, and X 10 is selected from K or R, X 11 is selected from A or V, X 12 is selected from K or T, X 13 It is selected from S or A, X 14 is selected from T or R, X 15 It is selected from S or T, X 16 The answer is selected from A or Q.

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

[0159] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 NOs. 7, 15, 23, 31, 39, 62, 63, 64, 65, 66, or 68, and a light chain variable region whose amino acid sequence 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 NOs. 8, 16, 24, 32, 40, 67, or 69.

[0160] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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, and a light chain variable region whose amino acid sequence 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 includes 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.

[0161] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 moiety further includes 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.

[0162] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 includes 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.

[0163] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 includes 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 region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 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.

[0165] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 includes 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.

[0166] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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, and a light chain variable region whose amino acid sequence 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 includes 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.

[0167] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 includes 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.

[0168] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 includes 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.

[0169] In some embodiments, the first antigen-binding region is a heavy chain variable region whose amino acid sequence 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: 66, and the amino acid sequence is at least 85%, 86%, 87%, 8 Includes a light chain variable region having identity of 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, where 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 is selected from K or R, X 11 is selected from A or V, X 12 is selected from K or T, X 13 It is selected from S or A, X 14 is selected from T or R, X 15 It is selected from S or T, X 16 X1 is selected from A or Q. Furthermore, in some such embodiments, the first antigen-binding moiety 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: 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: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6, where X1 is selected from D or E and X2 is selected from G or A.

[0170] In some embodiments, the first antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 first 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0193] In this disclosure, the first antigen-binding moiety is a mouse antibody, a chimeric antibody, or a humanized antibody. Humanization can reduce immunogenicity. Therefore, in some embodiments, the first antigen-binding moiety 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 moiety of the bispecific antibody is selected from six CDRs of any exemplary monospecific antibody that binds to GPRC5D. For example, in some embodiments, the variable region of the first antigen-binding moiety of the bispecific antibody is selected from the heavy chain variable region and light chain variable region of any exemplary monospecific antibody that binds to GPRC5D.

[0195] In the bispecific antibodies of this disclosure, the second antigen-binding moiety provides the ability to target CD3. The second antigen-binding moiety may be a Fab, scFv, or scFab (single-stranded Fab). In some embodiments, the second antigen-binding moiety is a CD3-binding scFv.

[0196] In some embodiments, the second antigen-binding moiety is a mouse antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the second antigen-binding moiety is humanized. In some specific embodiments, both the first and second antigen-binding moieties are humanized.

[0197] In some embodiments, the second antigen-binding portion includes 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 region includes HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 100, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 101.

[0199] In some embodiments, the second antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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. In some embodiments, the second antigen-binding region includes 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 region includes 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 antibodies of this disclosure can bind to GPRC5D. In some embodiments, the bispecific antibodies can bind to human GPRC5D and / or monkey GPRC5D and / or mouse GPRC5D. In some embodiments, the bispecific antibodies do not bind to, or substantially do not bind to, GPRC5A, GPRC5B, and GPRC5C.

[0201] In some specific examples, the bispecific antibodies of this disclosure may be GC35 or GC39.

[0202] The bispecific antibodies of this disclosure can target GPRC5D expressed on the surface of tumor cells and can also bind to T cells to induce T cell activation and activate the tumor-killing activity of T cells. The bispecific antibodies of this disclosure exhibit excellent antitumor properties, such as lysing tumor cells or inhibiting tumor cell proliferation. In some embodiments, the bispecific antibodies exhibit excellent antitumor properties against tumors such as multiple myeloma.

[0203] The molecular design of the bispecific antibodies described herein facilitates expression and purification.

[0204] The bispecific antibodies of this disclosure have good safety.

[0205] Composition of bispecificity antibodies The bispecific antibodies of this disclosure may not have an Fc domain, and the first antigen-binding portion and the second antigen-binding portion may be fused via an appropriate linker.

[0206] The bispecific antibodies of this disclosure may have an Fc domain that can extend the half-life and provide functions such as effectors associated with the Fc domain. In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety of the bispecific antibodies of this disclosure may be fused to the N-terminus of the Fc domain.

[0207] In the bispecific antibodies of the present disclosure, the first antigen-binding portion and the second antigen-binding portion can take any suitable structural form. In some embodiments, the first antigen-binding portion and the second antigen-binding portion can each independently be a Fab, scFv, scFab, or other structure.

[0208] The bispecific antibodies of the present disclosure can be multivalent, for example, bivalent, tetravalent, etc. In some embodiments, the bispecific antibody is bivalent, that is, the first antigen-binding portion and the second antigen-binding portion each provide a monovalent binding to the corresponding antigen.

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

[0210] In some embodiments, the bispecific antibodies of the present disclosure further include an Fc domain consisting of two Fc polypeptides.

[0211] In some embodiments, the first antigen-binding portion is a Fab, the second antigen-binding portion is an scFv, the C-terminus of the Fab heavy chain of the first antigen-binding portion is fused to the N-terminus of one of the Fc polypeptides of the Fc domain, and the C-terminus of the second antigen-binding portion is fused to the N-terminus of the other Fc polypeptide of the Fc domain. Such a configuration is schematically shown in FIG. 9. In a more specific embodiment of this embodiment, the bispecific antibody is composed of three polypeptide chains, the first polypeptide chain includes the Fab light chain of the first antigen-binding portion, the second polypeptide chain includes the Fab heavy chain of the first antigen-binding portion and one of the Fc polypeptides of the Fc domain, and the third polypeptide chain includes the second antigen-binding portion and the other Fc polypeptide of the Fc domain.

[0212] In the bispecific antibody of this disclosure, 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 may be any suitable linker, such as a charged linker and / or a flexible linker.

[0213] In some embodiments, the second antigen-binding moiety is scFv, and the N-terminus-C-terminus structure is VH-VL. In some embodiments, the second antigen-binding moiety is scFv, and the N-terminus-C-terminus structure is VL-VH. 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, and these amino acids may be selected from 20 natural amino acids. In more preferred embodiments, the 1 to 50 amino acids are selected from glycine, alanine, proline, serine, asparagine, glutamine, and lysine. Thus, exemplary peptide linkers include polyglycine (particularly (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.

[0215] In some embodiments, the peptide linker may consist of glycine and serine. In some embodiments, the number of amino acids contained in the peptide linker may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20 or more. In some embodiments, the peptide linker is a peptide linker containing GGGGS as a unit. In some embodiments, the peptide linker with GGGGS as a unit is (GGGGS) nHerein, n is any number from 1 to 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, e.g., 1 to 5, 2 to 5, 3 to 6, 2 to 4, 1 to 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 embodiments, the first antigen-binding portion is Fab, the second antigen-binding portion is scFv, the C-terminus of the Fab heavy chain of the first antigen-binding portion is fused to the N-terminus of one Fc polypeptide of the Fc domain, the C-terminus of the heavy chain variable region of the second antigen-binding portion is fused to the N-terminus of the other Fc polypeptide of the Fc domain, and the C-terminus of the light chain variable region of the second antigen-binding portion is fused to the heavy chain variable region of the second antigen-binding portion, preferably 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 (GGGGS) 3.

[0217] When fused to an Fc domain, the fusion typically occurs via a hinge region. In one embodiment, the first antigen-binding moiety is fused to one Fc polypeptide of the Fc domain via a first hinge, and the second antigen-binding moiety is fused to the other Fc polypeptide of the Fc domain via a second hinge. In some embodiments, the first and second hinges can form a covalent bond, such as a disulfide bond, with each other. The first and / or second hinges may contain amino acids derived from the hinge region of human IgG, and the hinge region of human IgG may contain the native hinge region or its variants. In some embodiments, the first and / or second hinges contain amino acids derived from the hinge region of human IgG1. In some embodiments, the first and / or second hinges contain amino acids derived from the hinge region of human IgG4. In some specific embodiments, the first hinge includes EPKSCDKTHTCPPCP (SEQ ID NO: 102), and the second hinge includes GEPKSSDKTHTCPPCP (SEQ ID NO: 103).

[0218] In other embodiments, the first antigen-binding portion is Fab, and the second antigen-binding portion is scFv, wherein the C-terminus of the Fab light chain of the first antigen-binding portion is fused to the N-terminus of one of the Fc polypeptides of the Fc domain, and the C-terminus of the second antigen-binding portion is fused to the N-terminus of the other Fc polypeptide of the Fc domain.

[0219] FC Domain The Fc domain of a bispecific antibody is composed of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each Fc polypeptide contains the CH2 and CH3 regions 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 contains one 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 includes modifications such as amino acid substitutions. Such modifications may, for example, promote heterodimerization, alter effector function, or change the ability to bind to protein A.

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

[0223] The bispecific antibodies of this disclosure contain different antigen-binding moieties fused to one or the other of two Fc polypeptides in an Fc domain. Thus, the two Fc polypeptides typically comprise two different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization yield several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antibodies produced by recombination, it is advantageous to introduce modifications into the Fc domain of the bispecific antibody that promote the binding of the desired polypeptide. Therefore, in certain embodiments, the Fc domain includes amino acid substitutions that promote the association of the two Fc polypeptides within the Fc domain.

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

[0225] In certain embodiments, this modification is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two Fc polypeptides in the Fc domain and a "hole" modification on the other of the two Fc polypeptides in the Fc domain. Generally, this approach involves introducing a projection ("knob") at the interface of one Fc polypeptide and a corresponding recess ("hole") at the interface of the other Fc polypeptide, thereby promoting heterodimer formation and inhibiting homodimer formation by positioning the projection in the recess. The projection is constructed by substituting a smaller amino acid side chain (e.g., tyrosine or tryptophan) at the interface of one Fc polypeptide. At the interface of the other Fc polypeptide, a complementary recess of the same or similar size as the projection is formed by substituting a larger amino acid side chain (e.g., alanine or threonine) for a smaller amino acid side chain.

[0226] Therefore, in certain embodiments, a protrusion is formed within the CH3 domain of one Fc polypeptide of the bispecific antibody by substituting an amino acid residue in the CH3 domain of the bispecific antibody with an amino acid residue having a larger side chain volume, and this protrusion can be located within a recess in the CH3 domain of the other Fc polypeptide, and in the CH3 domain of the other Fc polypeptide, a recess is formed within the CH3 domain of the Fc polypeptide by substituting an amino acid residue with an amino acid residue having a smaller side chain volume.

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

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

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

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

[0231] Amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or impair its effector function are present in both polypeptide chains of the Fc domain.

[0232] In some embodiments, the Fc domain includes modifications that reduce or eliminate the binding of one Fc polypeptide's CH3 region within the Fc domain to protein A (derived from Staphylococcus aureus). In some embodiments, the Fc domain includes amino acid substitutions that reduce or eliminate the binding of one Fc polypeptide's CH3 region within the Fc domain to protein A. In some embodiments, according to EU numbering, the Fc domain includes amino acid substitutions (a) 435R or (b) 435R and 436F present in only one of the Fc polypeptides but not in the other. In some embodiments, according to EU numbering, the Fc domain includes amino acid substitutions (a) 435R or (b) 435R and 436F present in only one of the Fc polypeptides. In some specific embodiments, according to EU numbering, the Fc domain includes amino acid substitutions H435R and Y436F occurring in only one of the Fc polypeptides. In some specific embodiments, according to EU numbering, the Fc domain includes amino acid substitution H435R occurring in only one of the Fc polypeptides. In the above embodiment, this Fc domain is IgG1 Fc, and more particularly, human IgG1 Fc.

[0233] In the bispecific antibodies of this disclosure, the Fc domain may include one, two, or three modifications that promote heterodimerization, modify effector function, and reduce or eliminate binding between the CH3 region of one Fc polypeptide in the Fc domain and protein A. In some embodiments, the Fc domain includes modifications that promote heterodimerization, modify effector function, and reduce or eliminate binding between the CH3 region of one Fc polypeptide in the Fc domain and protein A. Specific forms of the different types of modifications described above may be combined. For example, in a particular embodiment, according to EU numbering, the Fc domain includes the following amino acid substitutions: i. 349C, 366S, 368A, 407T / V, 354C, and 366Y / W (wherein amino acid substitutions 354C and 366Y / W are present in the same Fc polypeptide, but not in 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 (present in only one of the Fc polypeptides).

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

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

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

[0237] In some embodiments, the bispecific antibody is composed of three polypeptide chains, one polypeptide chain containing 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, and another polypeptide chain containing at least 85%, 80% identity with the amino acid sequence shown in SEQ ID NO: 78. The polypeptide chain comprises an amino acid sequence having 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and further comprises 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 is composed of three polypeptide chains, one polypeptide chain containing 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, and another polypeptide chain containing at least 85%, 80% identity with the amino acid sequence shown in SEQ ID NO: 82. The polypeptide chain comprises an amino acid sequence having 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and further comprises 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 is composed of three polypeptide chains, one polypeptide chain containing the amino acid sequence shown in SEQ ID NO: 84, another polypeptide chain containing the amino acid sequence shown in SEQ ID NO: 78, and yet another polypeptide chain containing the amino acid sequence shown in SEQ ID NO: 88.

[0240] In some embodiments, the bispecific antibody is composed of three polypeptide chains, one polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 86, another polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 82, and yet another polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 88.

[0241] This disclosure provides exemplary bivalent bispecific antibodies.

[0242] In one example, a bispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are shown in SEQ ID NOs. 84, 78, and 88, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are shown in SEQ ID NOs. 85, 79, and 89, respectively.

[0243] In one example, a bispecific antibody is composed of three polypeptide chains, the amino acid sequences of the three polypeptide chains are shown in SEQ ID NOs. 86, 82, and 88, respectively. In some embodiments, the nucleotide sequences encoding the three polypeptide chains are shown in SEQ ID NOs. 87, 83, and 89, respectively.

[0244] Isolated nucleic acids This disclosure provides isolated nucleic acids comprising nucleotide sequences encoding bispecific antibodies described herein. The sequence listing exemplifies several nucleotide sequences encoding bispecific antibodies.

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

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

[0247] Pharmaceutical composition This disclosure provides a pharmaceutical composition comprising the bispecific antibody of this disclosure and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include, for example, excipients, diluents, encapsulants, fillers, buffers, or other agents.

[0248] Method for preparing bispecific antibodies In some embodiments, the present disclosure provides a method for preparing bispecific antibodies, the method comprising culturing host cells to express bispecific antibodies, and isolating and purifying the bispecific antibodies in system. To produce the bispecific antibodies, the nucleic acids encoding the bispecific antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be obtained using various methods 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, and ceramic hydroxyapatite (CHT) chromatography. The actual conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, which are apparent to those skilled in the art. In affinity chromatography, the bispecific antibody can be purified using an antibody, ligand, receptor, or antigen that binds to the bispecific antibody. For example, affinity chromatography of the bispecific antibody of this disclosure can be performed using a matrix containing protein A or protein G. The bispecific antibody of this disclosure can be sequentially purified using affinity chromatography, ion-exchange chromatography, gel chromatography, and / or CHT chromatography.

[0250] The purity of bispecific antibodies can be determined by any of the following well-known analytical methods: gel electrophoresis, high-performance liquid chromatography, size exclusion chromatography, etc.

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

[0252] use This disclosure provides the use of bispecific antibodies as described herein. In some specific embodiments, the bispecific antibodies 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 drug for treating a disease expressing GPRC5D. In some embodiments, the bispecific antibody or the pharmaceutical composition is combined with one or more additional therapeutic agents to prepare a drug. The additional therapeutic agents may be oncological agents or autoimmune disease agents known in the art.

[0254] This disclosure provides a method for treating a disease expressing GPRC5D, the method comprising administering the bispecific antibody or the pharmaceutical composition to a subject in need. This disclosure provides a method for treating a disease expressing GPRC5D, comprising administering a therapeutically effective amount of 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 hematological malignancy. In some embodiments, the tumor is a B-cell lymphoma. In some embodiments, the tumor is a B-cell lymphoma expressing GPRC5D. In some embodiments, the tumor is multiple myeloma (MM). In some embodiments, the tumor is a multiple myeloma expressing GPRC5D.

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

[0257] Specific Embodiments This disclosure also provides the following specific embodiments, but the scope of protection provided by this disclosure is not limited to these.

[0258] Embodiment 1. A bispecific antibody comprising a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety 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.

[0259] Embodiment 2. The first antigen-binding portion is (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 (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: 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 A bispecific antibody according to Embodiment 1, comprising:

[0260] Embodiment 3. A bispecific antibody comprising a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NOs. 7, 15, 23, 31, 39, 66, or 68, and LCDR1, LCDR2, and LCDR3 in a light chain variable region consisting of the amino acid sequence shown in SEQ ID NOs. 8, 16, 24, 32, 40, 67, or 69.

[0261] Embodiment 4. The first antigen-binding portion is (1) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8; (2) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16; (3) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24; (4) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 31, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 32; (5) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 39, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40; (6) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 62, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (7) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (8) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 64, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (9) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 65, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67; (10) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 66, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67 (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 is selected from K or R, X 11 is selected from A or V, X 12 is selected from K or T, X 13 It is selected from S or A, X 14 is selected from T or R, X 15 It is selected from S or T, X 16 is selected from A or Q); or (11) HCDR1, HCDR2, and HCDR3 in the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 68, and LCDR1, LCDR2, and LCDR3 in the light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 69 A bispecific antibody according to Embodiment 3, comprising:

[0262] Embodiment 5. A bispecific antibody according to any one of Embodiments 1 to 4, wherein the first antigen-binding portion includes a heavy chain variable region whose amino acid sequence 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 NOs: 7, 15, 23, 31, 39, 62, 63, 64, 65, 66, or 68, and a light chain variable region whose amino acid sequence 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 NOs: 8, 16, 24, 32, 40, 67, or 69.

[0263] Embodiment 6. The first antigen-binding portion is (1) A heavy chain variable region whose amino acid sequence 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; and a light chain variable region whose amino acid sequence 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 whose amino acid sequence 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 whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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) A heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 whose amino acid sequence 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; and a light chain variable region whose amino acid sequence 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; (6) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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; (7) Heavy chain variable regions whose amino acid sequence 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, and light chain variable regions whose amino acid sequence 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) A heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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; (10) A heavy chain variable region whose amino acid sequence 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: 66, and a light chain variable region whose amino acid sequence 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; or (11) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 A bispecific antibody according to any one of Embodiments 1 to 5, including the above.

[0264] Embodiment 7. The first antigen-binding portion is (1) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 8; (2) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 16; (3) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 24; (4) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 32; (5) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 40; (6) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (7) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (8) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (9) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (10) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; or (11) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68, and light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69 A bispecific antibody according to any one of Embodiments 1 to 6, including the above.

[0265] Embodiment 8. The bispecific antibody according to Embodiment 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.

[0266] Embodiment 9. The bispecific antibody according to Embodiment 8, wherein the first antigen-binding portion comprises a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 preferably the first antigen-binding portion comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67.

[0267] Embodiment 10. The bispecific antibody according to Embodiment 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.

[0268] Embodiment 11. The bispecific antibody according to Embodiment 10, wherein the first antigen-binding portion comprises a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 preferably the first antigen-binding portion comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69.

[0269] Embodiment 12. A bispecific antibody according to any one of Embodiments 1 to 11, wherein 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.

[0270] Embodiment 13. A bispecific antibody according to any one of Embodiments 1 to 11, wherein the second antigen-binding portion comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 100, and LCDR1, LCDR2, and LCDR3 in a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 101.

[0271] Embodiment 14. A bispecific antibody according to any one of Embodiments 1 to 13, wherein the second antigen-binding portion includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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.

[0272] Embodiment 15. A bispecific antibody according to any one of Embodiments 1 to 13, wherein the second antigen-binding portion includes 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.

[0273] Embodiment 16. A bispecific antibody according to any one of Embodiments 1 to 15, wherein the first antigen-binding moiety is mouse, chimeric, or humanized.

[0274] Embodiment 17. A bispecific antibody according to any one of Embodiments 1 to 16, wherein the second antigen-binding moiety is mouse, chimeric, or humanized.

[0275] Embodiment 18. A bispecific antibody according to any one of Embodiments 1 to 17, wherein the first antigen-binding portion and the second antigen-binding portion are each independently Fab, scFv, or scFab.

[0276] Embodiment 19. The bispecific antibody according to Embodiment 18, wherein the first antigen-binding portion is Fab and the second antigen-binding portion is scFv.

[0277] Embodiment 20. The bispecific antibody according to any one of Embodiments 1 to 19, wherein the bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.

[0278] Embodiment 21. The bispecific antibody according to Embodiment 20, wherein the first antigen-binding portion is Fab, the second antigen-binding portion is scFv, the C-terminus of the Fab heavy chain of the first antigen-binding portion is fused to the N-terminus of one Fc polypeptide of the Fc domain, and the C-terminus of the second antigen-binding portion is fused to the N-terminus of the other Fc polypeptide of the Fc domain.

[0279] Embodiment 22. The bispecific antibody according to Embodiment 21, wherein the N-terminus to C-terminus structure of the second antigen-binding moiety is VH-VL or VL-VH, and 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, preferably the peptide linker is (GGGGS)3.

[0280] Embodiment 23. A bispecific antibody according to any one of Embodiments 20 to 22, wherein the Fc domain is an IgG Fc domain, preferably an IgG1 Fc domain.

[0281] Embodiment 24. The bispecific antibody according to Embodiment 23, wherein the IgG Fc domain is a human IgG Fc domain, preferably a human IgG1 Fc domain.

[0282] Embodiment 25. A bispecific antibody according to any one of Embodiments 20 to 24, wherein the Fc domain includes an amino acid substitution that promotes the association of two Fc polypeptides within the Fc domain.

[0283] Embodiment 26. The bispecific antibody according to Embodiment 25, wherein, according to EU numbering, one Fc polypeptide 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.

[0284] Embodiment 27. A bispecific antibody according to any one of Embodiments 20 to 26, wherein the Fc domain includes an amino acid substitution that reduces the binding affinity of the Fc domain to the Fc receptor and / or reduces its effector function.

[0285] Embodiment 28. A bispecific antibody according to Embodiment 27, wherein, according to EU numbering, an amino acid substitution that reduces the binding affinity of the Fc domain to the Fc receptor and / or reduces its effector function is located at one or more positions selected from 234, 235, and 329, preferably, both of the two Fc polypeptides of the Fc domain contain amino acid substitutions 234A, 235A, and 329G, or both contain amino acid substitutions 234A, 235A, and 329A.

[0286] Embodiment 29. A bispecific antibody according to any one of Embodiments 20 to 28, wherein the Fc domain includes an amino acid substitution that reduces or eliminates the binding of the CH3 region of one Fc polypeptide in the Fc domain to protein A.

[0287] Embodiment 30. A bispecific antibody according to Embodiment 29, wherein the Fc domain comprises an amino acid substitution (a) 435R, or (b) 435R and 436F, which is present in only one of the Fc polypeptides, according to EU numbering.

[0288] Embodiment 31. A bispecific antibody according to any one of Embodiments 20 to 24, wherein one Fc polypeptide of the Fc domain comprises the following amino acid substitutions: L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide comprises the following amino acid substitutions: (a) L234A, L235A, P329A, S354C, T366W, and H435R, or (b) L234A, L235A, P329A, S354C, T366W, H435R, and Y436F.

[0289] Embodiment 32. The bispecific antibody according to any one of Embodiments 1 to 31, wherein the bispecific antibody is divalent.

[0290] Embodiment 33. The bispecific antibody is composed of three polypeptide chains, (1) 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: 84, and the other polypeptide chain contains at least 85%, 86%, 87%, 88%, 89%, 90%, 90% identical to the amino acid sequence shown in SEQ ID NO: 78. The polypeptide chain contains an amino acid sequence having 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 88, or the 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, or (2) 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, and the other polypeptide chain contains at least 85%, 86%, 87%, 88%, 89%, or 90% identity with the amino acid sequence shown in SEQ ID NO: 82. , comprising an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and further comprising one polypeptide chain 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, A bispecific antibody according to any one of Embodiments 1 to 32.

[0291] Embodiment 34. Composed of three polypeptide chains, (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 yet another 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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 88. Bispecific antibodies.

[0292] Embodiment 35. An isolated nucleic acid comprising a nucleotide sequence encoding a bispecific antibody as described in any one of Embodiments 1 to 34.

[0293] Embodiment 36. A vector comprising the nucleic acid described in Embodiment 35.

[0294] Embodiment 37. A host cell comprising the nucleic acid described in Embodiment 35 or the vector described in Embodiment 36.

[0295] Embodiment 38. A method for preparing a bispecific antibody according to any one of Embodiments 1 to 34, comprising culturing the host cells described in Embodiment 37 to express the bispecific antibody, and isolating and purifying the bispecific antibody in system.

[0296] Embodiment 39. A pharmaceutical composition comprising a bispecific antibody according to any one of Embodiments 1 to 34 and a pharmaceutically acceptable additive.

[0297] Embodiment 40. Use of a bispecific antibody according to any one of Embodiments 1 to 34 or a pharmaceutical composition according to Embodiment 39 in the preparation of a drug for treating a disease expressing GPRC5D.

[0298] Embodiment 41. The use according to Embodiment 40, wherein the disease is an autoimmune disease or a tumor, and the tumor is preferably a non-solid tumor, more preferably multiple myeloma.

[0299] Embodiment 42. The use according to Embodiment 40 or 41, wherein the bispecific antibody or the pharmaceutical composition is combined with one or more additional therapeutic agents to prepare the drug.

[0300] Embodiment 43. A method for treating a disease expressing GPRC5D, comprising administering a therapeutically effective amount of a bispecific antibody according to any one of Embodiments 1 to 34 or a pharmaceutical composition according to Embodiment 39 to a subject in need thereof.

[0301] Embodiment 44. The method according to Embodiment 43, wherein the disease is an autoimmune disease or a tumor, and the tumor is preferably a non-solid tumor, more preferably multiple myeloma.

[0302] Embodiment 45. A monospecific antibody or its antigen-binding moiety that binds to GPRC5D, comprising a heavy chain variable region whose amino acid sequence 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, 63, 64, 65, 66, or 68, and a light chain variable region whose amino acid sequence 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 or 69.

[0303] Embodiment 46.(1) A heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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 whose amino acid sequence 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, and a light chain variable region whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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; (4) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 whose amino acid sequence 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: 66, and a light chain variable region whose amino acid sequence 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; or (6) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 A monospecific antibody or its antigen-binding moiety according to Embodiment 45, including the above.

[0304] Embodiment 47.(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 containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (3) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (4) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; or (5) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68, and light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69 A monospecific antibody or its antigen-binding moiety according to Embodiment 45 or 46, including the above.

[0305] Embodiment 48. An antibody-drug conjugate comprising a monospecific antibody or its antigen-binding moiety as described in any one of Embodiments 45 to 47.

[0306] Embodiment 49. An anti-GPRC5D chimeric antigen receptor comprising a monospecific antibody or its antigen-binding moiety as described in any one of Embodiments 45 to 47.

[0307] Embodiment 50. Manipulated immune effector cells containing the anti-GPRC5D chimeric antigen receptor described in Embodiment 49.

[0308] For clarity, the present disclosure will be further illustrated by examples, but these examples will not limit the scope of the present disclosure. The reagents used in the present disclosure are generally commercially available and can be used without further purification.

[0309] Example 1: Antigen Immunity 1. DNA antigen immunization The cDNA sequence encoding the human GPRC5D protein (amino acid sequence shown in SEQ ID NO: 41) was obtained by 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 for use with the endotoxin-free Max extraction kit (QIAGEN, catalog number: 12391).

[0310] Plasmid pcDNA3.1-hGPRC5D was used as an antigen to immunize A / J mice (Nanjing University Institute of Model Animals, MARC), BALB / c mice (Shanghai Lingchang), and SJL mice (Beijing Charles River). First, each mouse was pre-treated by pre-injecting hyaluronidase (Sigma, catalog number: H4272) into the muscles of its left and right hind limbs. Then, CpG (InvivoGen, catalog number: tlrl-1826) and plasmid pcDNA3.1-hGPRC5D were uniformly mixed in a mass ratio of 1:1. The uniformly mixed antigen complex was injected into the muscle tissue of the pre-treated mice via a bio-gene transduction device (Type II, Shanghai Teresa Bio-Tech Co., Ltd.), and the same immunization process was repeated every 2-3 weeks for a total of four immunizations. After completion of immunization, serum was collected from each mouse.

[0311] 2. Evaluation of mouse serum titers To evaluate the immune response to anti-human GPRC5D antibodies produced in mice immunized with DNA antigens, antibody titers in mouse serum were detected by FACS. Mice with higher serum titers were selected for subsequent shock immunization before splenocyte fusion.

[0312] 3. Cellular antigen immunity Following the instructions for use with the Lipofectamine 3000 transfection reagent (Thermo, catalog number: L3000015), plasmid pcDNA3.1-hGPRC5D was transfected into CHO-K1 cells to obtain a stable and highly expressive hGPRC5D cell line, CHO-K1-hGPRC5D. hiObtained the above CHO-K1-hGPRC5D hi Mice were subjected to shock immunization using cells as antigens. 3-4 days before spleen cell fusion, the final cell concentration was 5 × 10⁶. 7 CHO-K1-hGPRC5D cells / mL hi The cell suspension and CpG (InvivoGen, catalog number: tlrl-1826) at a final concentration of 0.5 mg / mL were homogeneously mixed, and then injected into the peritoneal cavity of mice at a rate of 100 μL per mouse.

[0313] Example 2: Screening of mouse anti-human GPRC5D antibody 1. Preparation of hybridoma cells On the day of fusion, mouse spleens were aseptically removed, pulverized, and washed with erythrocyte lysate (Sigma, catalog number: R7757) and PBS solution. The spleen cells were then resuspended in electrofusion buffer (BTX, catalog number: 47-0001), and the spleen cells and SP2 / 0 mouse myeloma cells were homogeneously mixed in a cell number ratio of 2:1. The spleen cells were fused 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 hybridoma cells were prepared by culturing at 37°C and 5% CO2 for 7-10 days. The culture supernatant of the hybridoma cells was collected and used for screening for mouse anti-human GPRC5D antibodies.

[0314] 2. Construction of stable cell lines To evaluate the interspecies cross-binding activity of mouse anti-human GPRC5D antibodies at the cellular level, the following cell lines were constructed. Full-length cDNA encoding monkey GPRC5D (amino acid sequence shown in SEQ ID NO: 42) and mouse GPRC5D (amino acid sequence shown in SEQ ID NO: 43) was obtained by gene synthesis, and subcloned into the vector pcDNA3.1(+) to obtain recombinant plasmids pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D. Following the instructions for use of the lipofectamine 3000 transfection reagent (Thermo, catalog number: L3000015), the plasmids pcDNA3.1-cynoGPRC5D and pcDNA3.1-murineGPRC5D were transfected into CHO-K1 cells, respectively, to obtain stable cell lines CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D.

[0315] 3. Preliminary cell ELISA screening CHO-K1-hGPRC5D during logarithmic growth phase hi The cells were harvested and resuspended in DMEM / F-12 (Gibco, catalog number: 11320033) medium containing 10% by volume of FBS, and the cell concentration was increased to 1 × 10⁶. 6The cells were adjusted to the required concentration per mL and plated into a 96-well flat-bottom plate at 100 μL / well. The cells were incubated overnight at 37°C under 5% CO2 conditions. The following day, the cell plates were washed with PBS and fixed at room temperature for 30 minutes with 4% (vol. percent) paraformaldehyde (Beyotime, catalog number: P0099). After washing with PBS, 200 μL / well of PBS solution containing 3% (vol. percent) BSA was added, and the cells were blocked at room temperature for 2 hours. After blocking, 100 μL / well of hybridoma cell culture supernatant was added to the cell plates, and the cells 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 cells were incubated at room temperature for 1 hour. After washing with PBS, 100 μL / well of TMB reaction solution (Solarbio, catalog number: RP1200) was added, incubated at room temperature in the dark for 5 minutes, and the reaction was stopped with 0.5 M H2SO4. OD was performed using a Bio-rad iMark microplate reader. 450nm Absorbance values ​​were read. Hybridoma cells corresponding to culture supernatants with absorbances greater than 1.0 were selected as positive clones for subsequent FACS screening.

[0316] 4. Flow cytometry FACS screening of antibodies Cell concentration 5 × 10⁶ in a 96-well U-bottom plate 5CHO-K1-cynoGPRC5D cell suspension at a concentration of cells / mL was plated in 100 μL / wells, and the culture supernatant of positive clones was added in 100 μL / wells and mixed homogeneously, followed by incubation at 4°C for 1 hour. After washing with PBS containing 2% (vol. 1%) FBS, AlexaFluor488-conjugated goat anti-mouse IgG+IgM(H+L) antibody (Jackson Immuno Research, catalog number: 115-545-044) was added and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (vol. 1%) FBS and resuspended, and fluorescence signals were detected using a flow cytometer (Sartorius, IQue3). Binding of anti-hGPRC5D antibody to CHO-K1-cynoGPRC5D cells was analyzed by mean fluorescence intensity (MFI) of staining.

[0317] 5. Preparation of subcloning After screening by cell ELISA and FACS, CHO-K1-hGPRC5D hi Hybridoma cells positively binding to both CHO-K1-cynoGPRC5D were selected for proliferation and culture. The proliferated cells were homogeneously mixed with semi-solid medium D (Stemcell, catalog number: 3810) in a volume ratio of 1:10 and cultured for 5 days at 37°C and 5% CO2. Monoclonal cell clusters were aspirated under a 4x microscope and transferred to a 96-well plate covered with hybridoma medium (Gibco, catalog number: 12045-076), where they were cultured for 2 days at 37°C and 5% CO2. Subclonal cell culture supernatant was collected from each well and used for further screening of subclonal cells.

[0318] 6. Subclone screening (1) FACS screening of anti-hGPRC5D antibodies Cell concentration 3×10 5NCI-H929 cell suspension at a concentration of cells / mL was plated in 96-well U-bottom plates at 100 μL / well. Subclonal cell culture supernatant was added at 100 μL / well or an equivalent volume of medium, and after homogeneous mixing, the cells were incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (volume percent) FBS, AlexaFluor488-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% (volume percent) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3), and the binding of mouse anti-hGPRC5D antibody to NCI-H929 cells was analyzed by mean fluorescence intensity (MFI) of staining. Some results are shown in Table 1. [Table 2]

[0319] (2) Cell ELISA screening of anti-hGPRC5D antibody CHO-K1-hGPRC5D during logarithmic growth phase hi Cells, CHO-K1-cynoGPRC5D cells, CHO-K1-murineGPRC5D cells, and CHO-K1 cells were collected and resuspended in DMEM / F-12 (Gibco, catalog number: 11320033) medium containing 10% (volume percent) FBS, and the cell concentration was increased to 1 × 10⁻⁶ 6The cells were adjusted to the required concentration per mL and plated at 100 μL / well in a 96-well flat-bottom plate. The cells were incubated overnight at 37°C under 5% CO2 culture conditions. The following day, the cell plates were washed with PBS, 4% (vol. percent) paraformaldehyde (Beyotime, catalog number: P0099) was added, and the plates were fixed at room temperature for 30 minutes. After washing with PBS, 200 μL / well of PBS solution containing 3% (vol. percent) BSA was added, and the plates were blocked at room temperature for 2 hours. After blocking, 50 μL / well of subclonal cell culture supernatant was added to the cell plates, 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, 100 μL / well of TMB reaction solution (Solarbio, catalog number: RP1200) was added, incubated at room temperature in the dark for 5 minutes, and the reaction was stopped with 0.5 M H2SO4. OD was performed using a Bio-rad iMark microplate reader. 450nm Absorbance values ​​were read. As shown in Figure 1, 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, mu-183, etc.) are CHO-K1-hGPRC5D hi The absorbance values ​​for cells, CHO-K1-cynoGPRC5D cells, and / or CHO-K1-murineGPRC5D cells are at least twice as high as those for control cells, CHO-K1.

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

[0321] 2. Construction of a chimeric antibody expression vector Using chemical synthesis, the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the mouse anti-hGPRC5D antibody were ligated to the nucleotide sequences of the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively. A recombinant human-mouse chimeric antibody expression vector was constructed using a pcDNA3.1(+) vector and used for transfection 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. [Table 3]

[0322] 3. Construction of a positive reference antibody expression vector GC5B596 was selected as the reference antibody (monoclonal antibody, source WO2018017786A2). The nucleotide sequences of antibody VH (sequence number 44) and VL (sequence number 45) were obtained using chemical synthesis, and these were ligated to the nucleotide sequences of the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively. An expression vector expressing the positive reference antibody was constructed using a pcDNA3.1(+) vector and used for transfection and expression to prepare the positive reference antibody. The positive reference antibody (monoclonal antibody) is referred to as BM-mAb in this specification.

[0323] 4. Preparation of anti-hGPRC5D chimeric antibody and positive reference antibody Following the operating manual for the expiCHO expression system (Gibco, catalog number: A29129), anti-hGPRC5D antibody was transiently transfected and expressed. After transfection, expiCHO cells were cultured with shaking at 37°C and 8% CO2 for 7 days. The cell culture supernatant was collected, and the clarified supernatant was loaded onto a Protein A column (GEHealthcare, catalog number: 17-5474). The Protein A column was washed with PBS buffer at 10 times the column volume, and the IgG antibody bound to the Protein A column was eluted and collected by adding acetate buffer (300 mM acetate, pH 3.6). The collected IgG antibody component was replaced with PBS buffer using an ultrafiltration device (molecular weight cutoff 30 kDa, Millipore, catalog number: UFC903024) to obtain the anti-hGPRC5D antibody solution.

[0324] Example 4: Measurement of the binding activity of anti-hGPRC5D antibody against cells that stably express human GPRC5D. 1. Binding characteristics of anti-hGPRC5D chimeric antibodies against stable, high-expression human GPRC5D cells Cell concentration 3×10 5 CHO-K1-hGPRC5D cells / mL hi A cell suspension (GPRC5D expression level approximately 2E+06-3E+06 antigen / cell) was plated at 100 μL / well in a 96-well U-bottom plate. Gradient-diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold gradient dilution) was added, mixed uniformly, and incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (volume percent) 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. The cells were washed with PBS containing 2% (volume percent) FBS and resuspended, and fluorescence signals were detected using a flow cytometer (Sartorius, IQue3) to detect the presence of anti-hGPRC5D antibody and CHO-K1-hGPRC5D hi Cellular binding was analyzed by the mean fluorescence intensity (MFI) of the stain. The calculated EC 50The analysis results are shown in Figure 2. The chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, Chi-164, Chi-169, and Chi-180 all showed concentration gradient-dependent binding activity to cells highly expressing hGPRC5D and had a higher maximum antigen binding amount than BM-mAbs.

[0325] 2. Binding characteristics of anti-hGPRC5D chimeric antibodies to stable low-expression human GPRC5D cells Transfect CHO-K1 cells with plasmid pcDNA3.1-hGPRC5D according to the instructions for use with Lipofectamine 3000 Transfection Reagent (Thermo, Catalog No.: L3000015). low A stable, low-expression cell line (GPRC5D expression level approximately 2000-2500 antigens / cell) was obtained. Cell concentration 3 × 10⁻⁶ 5 CHO-K1-hGPRC5D cells / mL low The cell suspension was plated at 100 μL / well in a 96-well U-bottom plate, gradient-diluted anti-hGPRC5D antibody (final concentration range 4.1–3000 ng / mL, 3-fold gradient dilution) was added, mixed uniformly, and incubated at 4°C for 1 hour. After washing the cells with PBS containing 2% (vol. 1%) 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. The cells were washed with PBS containing 2% (vol. 1%) FBS and resuspended, and fluorescence signals were detected using a flow cytometer (Sartorius, IQue3) to detect the presence of anti-hGPRC5D antibody and CHO-K1-hGPRC5D low Cell binding was analyzed by mean fluorescence intensity (MFI) of staining. As shown in Figure 3, in cells with low hGPRC5D expression, the chimeric antibodies Chi-89, Chi-105, Chi-164, and Chi-180 showed binding levels comparable to BM-mAb, while Chi-128, Chi-131, and Chi-169 showed strong target binding ability.

[0326] Example 5: Measurement of the binding activity of anti-hGPRC5D antibody against tumor cells Cell concentration 3×10 5 NCI-H929 and MM.1S human myeloma cell suspensions at a concentration of cells / mL were plated in 100 μL / wells into 96-well U-bottom plates. Gradient-diluted anti-hGPRC5D antibody (final concentration range 4.1–1000 ng / mL, 3-fold gradient dilution) was added and mixed homogeneously, followed by incubation at 4°C for 1 hour. After washing the cells with PBS containing 2% (volume percent) 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. The cells were washed with PBS containing 2% (volume percent) 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) of staining. As can be seen from Figures 4A and 4B, the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, and Chi-169 all exhibit concentration-gradient binding activity to NCI-H929 cells and MM.1S cells.

[0327] Example 6: Seed cross-reactivity assay of anti-hGPRC5D antibody Cell concentration 5×10 5Cell suspensions of CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D at a concentration of cells / mL were plated at 100 μL / well in a 96-well U-bottom plate. Gradient-diluted anti-hGPRC5D antibody (final concentration range 4.6–10000 ng / mL, 3-fold gradient dilution) was added and mixed uniformly, then incubated at 4°C for 1 hour. After washing with PBS containing 2% (volume percent) 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 with PBS containing 2% (volume percent) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3), and the binding of anti-hGPRC5D antibodies to CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells was analyzed by mean fluorescence intensity (MFI) of staining. As shown in Figures 5A and 5B, the chimeric antibodies Chi-89, Chi-128, Chi-131, and Chi-169 all exhibited concentration-gradient binding activity to CHO-K1-cynoGPRC5D and CHO-K1-murineGPRC5D cells. This indicates that, compared to BM-mAbs, these anti-hGPRC5D antibodies simultaneously possess cross-binding activity with monkey and mouse GPRC5D, while the chimeric antibody Chi-105 possesses cross-binding activity only with monkey GPRC5D.

[0328] Example 7: Verification of the specificity of the anti-hGPRC5D antibody 1. Verification of the binding of anti-hGPRC5D chimeric antibodies to the same family of proteins at the cellular level. In addition to GPRC5D, known GPRC family proteins also include 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 obtained by gene synthesis and subcloned into the vector pcDNA3.1(+) to obtain recombinant plasmids pcDNA3.1-GPRC5A-GFP, pcDNA3.1-GPRC5B-GFP, and pcDNA3.1-GPRC5C-GFP. These recombinant plasmid DNAs were transfected into CHO-K1 cells according to the instructions for the Lipofectamine 3000 transfection reagent (Thermo, catalog number: L3000015) to obtain stable cell lines of CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C.

[0329] Cell concentration 5×10 5Cell suspensions of CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C at a concentration of cells / mL were plated in 96-well U-bottom plates at a rate of 100 μL / well. Gradient-diluted anti-hGPRC5D antibody (final concentration range 4.1-3000 ng / mL, 3-fold gradient dilution) was added and mixed uniformly, then incubated at 4°C for 1 hour. After washing with PBS containing 2% (volume percent) 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 with PBS containing 2% (volume percent) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3), and the binding of anti-hGPRC5D antibodies to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells was analyzed by mean fluorescence intensity (MFI) of staining. As shown in Figures 6A, 6B, and 6C, the chimeric antibodies Chi-89, Chi-105, Chi-128, Chi-131, and Chi-169 all did not bind to CHO-K1-GPRC5A, CHO-K1-GPRC5B, and CHO-K1-GPRC5C cells within the concentration range of 4.1–3000 ng / mL, while BM-mAb showed nonspecific binding to CHO-K1-GPRC5A cells at concentrations above 1000 ng / mL.

[0330] 2. Verification of the binding of anti-hGPRC5D chimeric antibody to CHO-K1 cells. First, biotin-conjugated anti-hGPRC5D antibodies were prepared according to the instructions for use of the EZ-linkNHS-Biotin reagent (Thermo, catalog number: 20217), and named Biotin-BM-mAb, Biotin-Chi-89, Biotin-Chi-128, and Biotin-Chi-131, respectively. Here, "Biotin" refers to biotin.

[0331] Cell concentration 3×10 5A CHO-K1 cell suspension of cells / mL was plated in 96-well U-bottom plates at 100 μL / well. Gradient-diluted biotin-conjugated anti-hGPRC5D antibody (3-fold gradient dilution, final concentration range 4.6–10000 ng / mL) was added and mixed homogeneously, then incubated at 4°C for 1 hour. After washing with PBS containing 2% (vol. 1%) FBS, PE-conjugated streptavidin (BD, catalog number: 554061) was added and incubated at 4°C for 1 hour. Cells were washed with PBS containing 2% (vol. 1%) FBS and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, IQue3), and the mean fluorescence intensity (MFI) of the stain was used to analyze whether the anti-hGPRC5D antibody, which showed amplified MFI signals after biotin conjugation, nonspecifically bound to CHO-K1 cells. As shown in Figure 7, the biotin-conjugated chimeric antibodies Chi-89, Chi-128, and Chi-131 all generated non-binding signals with CHO-K1 cells.

[0332] Example 8: Construction, expression, and purification of anti-GPRC5D humanized antibody The anti-GPRC5D chimeric antibodies Chi-131 and Chi-128 were humanized, respectively. After humanizing the chimeric antibody Chi-131, four humanized antibodies named hu131-v1, hu131-v2, hu131-v3, and hu131-v4 were obtained. After humanizing the chimeric antibody Chi-128, one humanized antibody named hu128-v1 was obtained. The amino acid and nucleotide sequences of the heavy and light chains of each humanized antibody are shown in Table 3. [Table 4]

[0333] The nucleotide sequences encoding the above-mentioned 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). The expression vectors containing the constructed nucleotide sequences were first transfected into ExpiCHO cells (CHO-S, Thermo Fisher Scientific). The cells were cultured in ExpiCHO expression medium on an orbital shaker platform at 130 rpm, 37°C, and in a humidified atmosphere containing 8% CO2. The culture supernatant was collected and purified using Protein A magnetic beads (Genscript, catalog number L00273). Protein concentrations were measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).

[0334] Example 9: Binding activity assay of anti-GPRC5D chimeric antibody and humanized antibody The binding activity of chimeric antibodies Chi-131 and Chi-128, as well as their humanized counterparts, to human GPRC5D was measured by binding to NCI-H929 cells (obtained from Nanjing Cobioer Biotechnology).

[0335] Cell concentration 1×10 6NCI-H929 human myeloma cell suspension at a concentration of cells / mL was seeded at 100 μL / well in 96-well U-bottom plates. Gradient-diluted anti-GPRC5D antibody (final concentration 310–1586 nM, 5-fold gradient dilution) was added, homogeneously mixed, and incubated at 4°C for 1 hour. After washing the cells with pre-cooled running buffer (MACS, catalog no. 130-091-221), PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog no. 109-116-170) was added, and incubated at 4°C for 0.5 hours. The cells were washed with pre-cooled running buffer and resuspended. Fluorescence signals were detected using a flow cytometer (Sartorius, iQue3). Binding of anti-hGPRC5D antibody to human myeloma cells was analyzed by mean fluorescence intensity (MFI) of staining. Figure 8 shows the binding activity of humanized antibodies (hu131-v1, hu131-v2, hu131-v3, hu131-v4, hu128-v1) and chimeric antibodies (Chi-131 and Chi-128) to the GPRC5D target antigen on NCI-H929 cells. All humanized antibodies (hu131-v1, hu131-v2, hu131-v3, hu131-v4, hu128-v1) and chimeric antibodies (Chi-131 and Chi-128) showed concentration-dependent binding activity to NCI-H929 cells.

[0336] Example 10: Construction, expression, and purification of anti-GPRC5D / anti-CD3 bispecific antibody (1) Anti-GPRC5D / anti-CD3 bispecific antibodies GC35 and GC39 The anti-GPRC5D / anti-CD3 bispecific antibodies GC35 and GC39 were constructed according to the configuration shown in Figure 9. Here, the first antigen-binding site is Fab, which binds to GPRC5D, and the second antigen-binding site is scFv, which 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.

[0337] Nucleotide sequences encoding anti-GPRC5D-HC, anti-GPRC5D-LC, and anti-CD3-scFv-Fc were synthesized and cloned into a pcDNA3.1(+) expression vector. The constructed expression vector was transfected using the ExpiCHO transfection kit (Thermo Fisher, catalog number A29133) at a ratio of vector anti-GPRC5D-HC:vector anti-GPRC5D-LC:vector anti-CD3-scFv-Fc = 1:1.5:1.5, with a transfection density of 6 × 10⁻¹⁶. 6 ExpiCHO cells were co-transfected with the antibody at a concentration of cells / mL. Cells were continuously cultured for 10–15 days post-transfection, and the supernatant was collected by centrifugation. Proteins were purified using the AEKTA pure protein purification system (GE Healthcare) by protein A affinity chromatography, ion exchange chromatography, and gel chromatography. Protein concentrations were measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific). Bispecific antibodies were confirmed to have the expected structure and sequence by electrophoresis (reduced SDS-PAGE), molecular weight analysis (TOF MS), and sequence analysis.

[0338] (2) Standard substance Talquetamab The anti-GPRC5D / anti-CD3 bispecific antibody Talquetamab was constructed as a standard (Benchmark, BM). Specifically, Talquetamab has four polypeptide chains, and their amino acid sequences are shown in Table 4-2. The nucleotide sequences encoding each polypeptide chain of Talquetamab were synthesized and cloned into a pcDNA3.1(+) expression vector. Using the ExpiCHO transfection kit (Thermo Fisher, A29133), the initial ExpiCHO was co-transfected with Talquetamab anti-GPRC5D-HC antibody and anti-GPRC5D-LC antibody in a vector anti-GPRC5D-HC:vector anti-GPRC5D-LC = 1:1.5 ratio to express an anti-GPRC5D monoclonal antibody. Talquetamab anti-CD3-HC and anti-CD3-LC were co-transfected into a second ExpiCHO in a vector anti-CD3-HC:vector anti-CD3-LC ratio of 1:1.5, and anti-CD3 monoclonal antibodies were expressed. Both ExpiCHOs expressed 6 × 10⁶ cells. 6 Cells were transfected at a density of cells / mL. The cells were continuously cultured for 10–15 days after transfection, and the supernatant was collected by centrifugation.

[0339] The supernatant was purified using the AEKTA Pure Protein Purification System (GE Healthcare). The purified anti-GPRC5D monoclonal antibody and anti-CD3 monoclonal antibody were assembled in vitro in equimolar quantities to produce Talquetamab. Specifically, the purified monoclonal antibody solution was replaced with PBS (pH 7.4) and concentrated. 2 mg of each monoclonal antibody was taken, totaling 4 mg (the molar ratio of anti-GPRC5D monoclonal antibody to anti-CD3 monoclonal antibody was approximately 1:1), and this was supplemented to 0.9 mL of PBS. 6 mg of 2-mercaptoethylamine·HCl (2-MEA, final concentration 50 mM) was dissolved in 100 μL of PBS and quickly added to the prepared antibody solution to create a 1 mL reaction system. The reaction mixture was thoroughly mixed and incubated at 37°C for 90 minutes. The reaction mixture was cooled to room temperature, concentrated by ultrafiltration to remove 2-MEA, and stored in PBS. After allowing the oxidation reaction to occur overnight at 4°C, the antibody concentration was measured. [Table 5] [Table 6]

[0340] Example 11: Conjugation of anti-GPRC5D / anti-CD3 bispecific antibody to multiple myeloma cells expressing GPRC5D. Using flow cytometry, we analyzed the binding of anti-GPRC5D / anti-CD3 bispecific antibodies (including Talquetamab, GC35, and GC39) to GPRC5D expressed on RPMI-8226 cells (obtained from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), MM.1S cells (obtained from Beina Biotech), and NCI-H929 cells (obtained from Nanjing Cobioer Biotechnology). hIgG1 monoclonal antibody (Biointron, catalog number B117901) was used as a negative control.

[0341] RPMI-8226, MM.1S, and NCI-H929 cells in the logarithmic growth phase were cultured in RPMI-1640 medium (Hyclone, catalog number SH30809.01) supplemented with 2% (v / v) FBS (fetal bovine serum), with a cell density of 1 × 10⁶ cells. 6 ~2×10 6 The solution was adjusted to cells / mL. This solution was seeded at 50 μL / well into a 96-well U-shaped cell culture plate (Costar, catalog no. 3799). Using this medium, anti-GPRC5D / anti-CD3 bispecific antibodies of various concentrations were prepared, with the highest antibody concentration being 200 nM. These were then sequentially diluted five-fold to produce a total of nine different concentrations. Different concentrations of antibody were added to each well of the 96-well plate, bringing the total volume to 50 μL per well. After homogeneous mixing, the mixture was incubated at 4°C for 1 hour. The cells were washed with pre-cooled running buffer (MACS, catalog no. 130-091-221), and the supernatant was discarded. Pre-cooled PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog no. 109-116-170) was added at 100 μL per well and resuspended. The mixture was thoroughly mixed and incubated at 4°C for 30 minutes. After washing, the cells were resuspended by adding 100 μL of pre-cooled running buffer per well, and after thorough mixing, flow cytometry was performed using Sartorius and iQue3, followed by data analysis.

[0342] Table 5 and Figures 10A-10C show the binding activity of anti-GPRC5D / anti-CD3 bispecific antibodies to RPMI-8226, MM.1S, and NCI-H929 cells. The results showed that GC35 and GC39 had stronger binding activity to the target cells RPMI-8226, MM.1S, and NCI-H929 than Talquetamab. [Table 7]

[0343] 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. An hIgG1 monoclonal antibody (Biointron, catalog number B117901) was used as a negative control.

[0344] Human PBMCs (peripheral blood mononuclear cells) are converted to CD3 using CD3 magnetic beads (Miltenyi, catalog number 130-097-043). + T cells were selected. Frozen PBMCs were thawed, washed twice by centrifugation, and then the cells were counted. Running buffer (MACS, catalog number 130-091-221) was added in 10 minutes. 7 Add 80 μL per cell, and then add 10 CD3 magnetic beads. 7 The solution was added at a rate of 20 μL per cell. The mixture was mixed and incubated at 4°C for 15 minutes. 10 7 The cells were washed with 1-2 mL of running buffer per cell. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in 500 μL of running buffer. The LS column (Miltenyi, catalog no. 130-042-401) was placed on the MidiMACS Starting Kit (LS) magnetic stand (Miltenyi, catalog no. 130-091-051), rinsed, and then washed three times with the cell suspension. The LS column was removed from the magnetic stand, placed in a clean centrifuge tube, and selected CD3 cells were collected by adding 5 mL of buffer. + T cells were collected.

[0345] After counting, use the running buffer to perform CD3 + T cell density 1 × 10 6 ~2×10 6Cells were adjusted to the required concentration per mL and seeded at 50 μL / well in a 96-well U-shaped cell culture plate (Costar, catalog number 3799). Using running buffer, anti-GPRC5D / anti-CD3 bispecific antibodies of various concentrations were prepared, with the highest antibody concentration being 200 nM. Ten different concentrations were prepared by sequentially diluting the antibody 5-fold. Different antibody concentrations were added to each well of the 96-well plate, bringing the total volume to 50 μL per well. After homogeneous mixing, the cells were incubated at 4°C for 1 hour. The cells were washed with pre-cooled running buffer (MACS, catalog number 130-091-221), and the supernatant was discarded. Pre-cooled PE-conjugated goat anti-human IgG Fcγ antibody (Jackson Immuno Research, catalog number 109-116-170) was added to each well at a rate of 100 μL and resuspended. The cells were mixed well and incubated at 4°C for 30 minutes. After washing, the cells were resuspended by adding 100 μL of pre-cooled running buffer per well, and after thorough mixing, flow cytometry was performed using Sartorius and iQue3, followed by data analysis.

[0346] Table 6 and Figure 11 show the CD3 of anti-GPRC5D / anti-CD3 bispecific antibodies. + This shows the ability to bind to T cells. From these results, it can be concluded that GC35 and GC39 are CD3 + It has been shown that it can bind to T cells. [Table 8]

[0347] Example 13: Cytotoxic activity of multiple myeloma target cells by anti-GPRC5D / anti-CD3 bispecific antibody T cells were prepared using human PBMCs, and the cytotoxic activity of anti-GPRC5D / anti-CD3 bispecific antibodies (including GC35, GC39, and Talquetamab) against target cells NCI-H929 (obtained from Nanjing Cobioer Biotechnology), MM.1S (obtained from Beina Biotech), and RPMI-8226 (obtained from the Cell Resources Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) was investigated. An hIgG1 monoclonal antibody (Biointron, catalog number B117901) was used as a negative control.

[0348] Target cells were cultured in RPMI-1640 medium (Hyclone, catalog number SH30809.01) containing 2% (v / v) FBS, resulting in 5 × 10⁶ cells. 5 The cell density was adjusted to cells / mL, and 50 μL / well was seeded into 96-well cell culture plates (Eppendorf, catalog number 0030730199). Using the above medium, anti-GPRC5D / anti-CD3 bispecific antibodies of various concentrations were prepared, with the highest antibody concentration being 10 nM. For NCI-H929 cells and MM.1S cells, the antibody was diluted 5-fold to prepare a total of 9 different concentrations. On the other hand, for RPMI-8226 cells, the antibody was diluted 6-fold to prepare a total of 8 different concentrations. Different concentrations of antibody were added to the 96-well plates, making a total of 50 μL per well. Human PBMC effector cells were cultured in the above medium at a concentration of 2.5 × 10⁶ 6Adjusted to a density of cells / mL, and 100 μL was added to each well. The drug administration group (50 μL of target cells + 100 μL of effector cells + 50 μL of antibody), the target cell group (50 μL of target cells + 150 μL of medium), the effector cell group (100 μL of human PBMC + 100 μL of medium), the target cell + effector cell group (50 μL of target cells + 100 μL of effector cells + 50 μL of medium), the blank control group (200 μL of medium), the lysis solution control group (200 μL of medium + 20 μL of lysis solution), and the target cell maximum release group (50 μL of target cells + 150 μL of medium + 20 μL of lysis solution) were set up such that the ratio of effector cells to target cells was 10:1 and incubated for 24 hours. To the target cell maximum release group and the lysis solution control group, a lysis solution (Promega, catalog number G182A) was added 45 minutes before the start of the assay. Cytotoxicity was detected using the CytoTox96 (registered trademark) Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). The absorbance at 490 nm was measured using a microplate reader, and the cell lysis rate was calculated. Cell lysis rate (%) = (OD 薬剤投与群 - OD 標的細胞群+エフェクター細胞群 ) / (OD 標的細胞最大放出群 - OD 標的細胞群 ) × 100%

[0349] Figures 12A - 12C and Table 7 show the lysis rates of the anti-GPRC5D / anti-CD3 bispecific antibody against various tumor cells. The anti-GPRC5D / anti-CD3 bispecific antibody GC35 showed excellent cytotoxic activity against NCI-H929 cells, MM.1S cells, and RPMI-8226 cells compared to GC39 and Talquetamab.

Table 9

[0350] Example 14: T cell activation by anti-GPRC5D / anti-CD3 bispecific antibody The ability of anti-GPRC5D / anti-CD3 bispecific antibodies (including GC35, GC39, and Talquetamab) to activate T cells in the presence of myeloma target cells was investigated by measuring the expression levels of T cell activation markers CD69 and CD25. T cell activation ability was evaluated based on the degree of concentration-dependent upregulation of these markers. An hIgG1 monoclonal antibody (Biointron, catalog number B117901) was used as a negative control.

[0351] Target cells MM.1S (obtained from Beina Biotech) were subjected to 5 × 10⁶ filtration in RPMI-1640 medium (Hyclone, catalog number SH30809.01) containing 2% (v / v) FBS. 5 The cell density was adjusted to cells / mL, and 50 μL / well was seeded into a 96-well cell culture plate (Eppendorf, catalog number 0030730199). Using the above medium, anti-GPRC5D / anti-CD3 bispecific antibodies of various concentrations were prepared, with the highest antibody concentration being 10 nM. The antibody was then diluted 5-fold to prepare a total of 8 different concentrations. Different concentrations of the antibody were added to the above 96-well cell culture plate, making a total of 50 μL per well. Human PBMCs, which are effector cells, were cultured using the above experimental medium in a manner of 2.5 × 10⁶ 6Adjusted to a density of cells / mL, and 100 μL was added to each well. The drug administration group (50 μL of target cells + 100 μL of effector cells + 50 μL of antibody), the target cell group (50 μL of target cells + 150 μL of medium), the effector cell group (100 μL of human PBMC + 100 μL of medium), the target cell + effector cell group (50 μL of target cells + 100 μL of effector cells + 50 μL of medium), and the blank control group (200 μL of medium) were set up such that the ratio of effector cells to target cells was 10:1, and co-incubated for 24 hours. The supernatant was removed by centrifugation, the cells were washed with pre-cooled running buffer (MACS, catalog number 130-091-221), and after discarding the supernatant, 100 μL of running buffer was added to each well and the cells were mixed. Next, 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) were added to each well, and incubated on ice for 30 minutes. The cells were washed with pre-cooled running buffer, and resuspended by adding 100 μL / well of running buffer. After thorough mixing, analysis was performed using a flow cytometer.

[0352] Figures 13A to 13D and Table 8 show the degree of T cell activation in the presence of MM.1S target cells. Here, CD25 / CD4 is the ratio of the number of CD25 + cells to the number of CD4 + cells, CD69 / CD4 is the ratio of the number of CD69 + cells to the number of CD4 + cells, CD25 / CD8 is the ratio of the number of CD25 + cells to the number of CD8 + cells, and CD69 / CD8 is the ratio of the number of CD69 + cells to the number of CD8 + cells. The significantly increased expression of CD25 and CD69 compared to the negative control indicates that the T cells are activated. Both GC35 and GC39 were able to effectively activate T cells.

Table 10

[0353] Example 15: Cytokine release induced by anti-GPRC5D / anti-CD3 bispecific antibody Cytokine release induced by anti-GPRC5D / anti-CD3 bispecific antibodies (including GC35 and Talquetamab) in the presence of target cells was investigated by measuring the content of IL-2, IL-6, TNF-α, and IFN-γ. The safety of the bispecific antibodies was evaluated based on the amount of cytokine release.

[0354] Target cells, NCI-H929 cells (obtained from Nanjing Cobioer Biotechnology), were subjected to 5 × 10⁶ filtration in RPMI-1640 medium (Hyclone, catalog number SH30809.01) containing 2% (v / v) FBS. 5 The cell density was adjusted to cells / mL, and 50 μL / well was seeded into a 96-well cell culture plate (Eppendorf, catalog number 0030730199). Using the above medium, anti-GPRC5D / anti-CD3 bispecific antibodies of various concentrations were prepared, with the highest antibody concentration being 10 nM. The antibody was then diluted 5-fold to prepare a total of 9 different concentrations. Different concentrations of antibody were added to the 96-well plate, making a total of 50 μL per well. Human PBMCs, which are effector cells, were cultured using the above experimental medium at a rate of 2.5 × 10⁶ 6The cells were adjusted to a density of cells / mL, and 100 μL was placed in each well. The drug administration group (50 μL target cells + 100 μL effector cells + 50 μL antibody), target cell group (50 μL target cells + 150 μL medium), effector cell group (100 μL human PBMC + 100 μL medium), target cell + effector cell group (50 μL target cells + 100 μL effector cells + 50 μL medium), effector cell + antibody group (100 μL effector cells + 50 μL antibody + 50 μL medium), and blank control group (200 μL medium) were set up so that the ratio of effector cells to target cells was 10:1, and incubated for 24 hours. The supernatant was collected by centrifugation, and the samples were processed according to the instructions of the Human IL-2 Precoated ELISA Kit (Tatsuyu, catalog number 1110203), Human IL-6 Precoated ELISA Kit (Tatsuyu, catalog number 1110603), Human TNF-α Precoated ELISA Kit (Tatsuyu, catalog number 1117203), and Human IFN-γ Precoated ELISA Kit (Tatsuyu, catalog number 1110003), and analyzed using a microplate reader (Infinite F50, TECAN).

[0355] Figures 14A-14D and Table 9 show GC35-induced cytokine release in the presence of NCI-H929 cells. [Table 11]

[0356] Example 16: Inhibition of NCI-H929 proliferation in a mouse tumor model using an anti-GPRC5D / anti-CD3 bispecific antibody. The tumor inhibitory effect of GC35 in a mouse xenograft model of NCI-H929 human multiple myeloma was investigated. NCI-H929 human myeloma cells were mixed with human PBMCs and suspended, and human PBMCs (0.5 × 10⁶) were used. 7 Cells / mL) and NCI-H929 cells (7.5 × 10⁶) 7A suspension of cells ( / mL) was obtained. Under sterile conditions, 0.1 mL of the suspension was inoculated into the right axilla of B-NDG mice (obtained from Biocytogen) per mouse. The diameter of the transplanted tumor was measured with calipers. The tumor was approximately 200 mm. 3 At the point of tumor proliferation, the mice were randomly divided into seven groups of six. The day of group division was designated as day 0 (D0). The treatment groups received the drug at a volume of 10 mL / kg via tail vein injection (iv), while the blank control groups received an equal volume of PBS solution on days 1 (D1), 4 (D4), 7 (D7), and 10 (D10), for a total of four doses. The antitumor effect of the antibody was dynamically monitored using tumor diameter measurement. Tumor volume was measured 2-3 times per week, and the body weight of the mice was measured and the data recorded. The overall behavior of the mice was observed and recorded daily.

[0357] The following formulas were used to calculate various indicators. Tumor volume (TV) (mm 3 ) = 1 / 2 × (a × b 2 (In the formula, a is the longest diameter of the tumor, and b is the shortest diameter of the tumor.) Relative tumor volume (RTV) = TV t / TV0(where TV0 is the tumor volume on day 0, TV t (This represents the tumor volume at each measurement.) Relative tumor growth rate (T / C)(%)=T RTV / C RTV ×100% (in the formula, T RTV This is the RTV of the treatment group, C RTV This is the RTV of the control group. Tumor suppression rate (TGI) (%) = (1 - tumor weight in the treatment group / tumor weight in the control group) × 100%.

[0358] The experimental results are shown in Figure 15 and Table 10. At high doses, Talquetamab and GC35 significantly inhibited the proliferation of human multiple myeloma cells NCI-H929 in subcutaneous transplant tumors, resulting in tumor regression. At medium and low doses, GC35 was more effective than Talquetamab. [Table 12]

[0359] The sequence information disclosed herein is summarized in Table S2 below. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6] [Table 13-7] [Table 13-8] [Table 13-9] [Table 13-10] [Table 13-11] [Table 13-12] [Table 13-13]

[0360] For explanatory and disclosure purposes, all patents, patent applications, and other specific publications are expressly incorporated herein by reference. These publications are provided solely for disclosure prior to the filing date of this disclosure. All statements regarding the dates or content of these documents are based on information available to the applicant and do not constitute any endorsement of the accuracy of the dates or content of these documents. Furthermore, any reference to these publications herein does not constitute an endorsement that such publications constitute common general knowledge in the art in any country.

[0361] Although this disclosure is described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that any modifications or improvements can be made to this disclosure. Therefore, any such modifications or improvements made without departing from the spirit of this disclosure will fall within the scope of the protections claimed by this disclosure.

Claims

1. A bispecific antibody comprising a first antigen-binding moiety that binds to GPRC5D and a second antigen-binding moiety that binds to CD3, wherein the first antigen-binding moiety 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 first antigen-binding portion is (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 (X) containing the amino acid sequence shown in SEQ ID NO: 6 １ is selected from D or E, X ２ (This 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: 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 A bispecific antibody according to claim 1, comprising:

3. The first antigen-binding portion includes a heavy chain variable region whose amino acid sequence 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 NOs. 62, 63, 64, 65, 66, 7, 15, 23, 31, 39, or 68, and a light chain variable region whose amino acid sequence 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 NOs. 67, 8, 16, 24, 32, 40, or 69, Preferably, the first antigen-binding portion is (1) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 8; and light chain variable regions whose amino acid sequence 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 Sequence ID No. 8; (2) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 40; and light chain variable regions whose amino acid sequence 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 Sequence ID No. 40; (6) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 62, and light chain variable regions whose amino acid sequence 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 Sequence ID No. 67; (7) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 63, and light chain variable regions whose amino acid sequence 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 Sequence ID No. 67; (8) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 64, and light chain variable regions whose amino acid sequence 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 Sequence ID No. 67; (9) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 65, and light chain variable regions whose amino acid sequence 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 Sequence ID No. 67; (10) A heavy chain variable region whose amino acid sequence 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: 66, and a light chain variable region whose amino acid sequence 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; or (11) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 Includes, More preferably, the first antigen-binding portion is (1) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 8; (2) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 16; (3) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 24; (4) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 32; (5) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 39, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 40; (6) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (7) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (8) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (9) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (10) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 66, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; or (11) Heavy chain variable region including the amino acid sequence shown in SEQ ID NO: 68, and light chain variable region including the amino acid sequence shown in SEQ ID NO: 69 A bispecific antibody according to claim 1 or 2, comprising:

4. The first antigen-binding portion includes 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. Preferably, the first antigen-binding portion includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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. Preferably, the bispecific antibody according to claim 1, wherein the first antigen-binding portion comprises a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:

67.

5. The bispecific antibody according to any one of claims 1 to 4, wherein 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.

6. The second antigen-binding region includes a heavy chain variable region whose amino acid sequence 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 whose amino acid sequence 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. Preferably, the bispecific antibody according to any one of claims 1 to 5, wherein the second antigen-binding portion includes 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.

7. The first antigen-binding site is a mouse antigen-binding site, a chimeric antigen-binding site, or a humanized antigen-binding site. The bispecific antibody according to any one of claims 1 to 6, wherein the second antigen-binding site is, in some cases, a mouse antigen-binding site, a chimeric antigen-binding site, or a humanized antigen-binding site.

8. The bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides, Preferably, the Fc domain is an IgG Fc domain, and more preferably an IgG1 Fc domain. Preferably, the IgG Fc domain is a human IgG Fc domain, and more preferably, a human IgG1 Fc domain. A bispecific antibody according to any one of claims 1 to 7.

9. The first antigen-binding portion is Fab, the second antigen-binding portion is scFv, the C-terminus of the Fab heavy chain of the first antigen-binding portion is fused to the N-terminus of one Fc polypeptide of the Fc domain, and the C-terminus of the second antigen-binding portion is fused to the N-terminus of the other Fc polypeptide of the Fc domain. In some cases, the N-terminus to C-terminus structure of the second antigen-binding moiety is VH-VL or VL-VH, and in some cases, 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, preferably the peptide linker is (GGGGS) ３ That is, The bispecific antibody according to claim 8.

10. The aforementioned FC domain: (1) Amino acid substitutions that facilitate the association between the two Fc polypeptides of the Fc domain; (2) Amino acid substitutions that reduce the binding affinity of the Fc domain to the Fc receptor and / or reduce its effector function; and / or (3) Amino acid substitutions that reduce or eliminate the binding between the CH3 region of one Fc polypeptide in the Fc domain and protein A. Includes, Preferably, according to EU numbering, one Fc polypeptide of the Fc domain contains amino acid substitutions L234A, L235A, P329A, Y349C, T366S, L368A, and Y407V, and the other Fc polypeptide contains amino acid substitutions (a) L234A, L235A, P329A, S354C, T366W, and H435R, or (b) L234A, L235A, P329A, S354C, T366W, H435R, and Y436F. The bispecific antibody according to claim 8 or 9.

11. The bispecific antibody is composed of three polypeptide chains, (1) 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: 84, and the other polypeptide chain contains at least 85%, 86%, 87%, 88%, 89%, 90%, 90% identical to the amino acid sequence shown in SEQ ID NO:

78. The polypeptide chain contains an amino acid sequence having 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 88, or the 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, 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: 86, and the other polypeptide chain contains at least 85%, 86%, 87%, 88%, 89%, or 90% identical to the amino acid sequence shown in SEQ ID NO:

82. , comprising an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, and further comprising one polypeptide chain 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, Preferably, the bispecific antibody is composed of three polypeptide chains. (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 yet another 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 yet another polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

88. A bispecific antibody according to any one of claims 1 to 10.

12. An isolated nucleic acid comprising a nucleotide sequence encoding a bispecific antibody according to any one of claims 1 to 11.

13. A host cell comprising the nucleic acid described in claim 12.

14. A method for preparing a bispecific antibody according to any one of claims 1 to 11, comprising culturing the host cells according to claim 13 to express the bispecific antibody, and isolating and purifying the bispecific antibody in system.

15. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 11 and a pharmaceutically acceptable additive.

16. A method for treating a disease exhibiting GPRC5D, comprising administering a therapeutically effective amount of a bispecific antibody according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 15 to a subject in need thereof, Preferably, the disease is an autoimmune disease or a tumor, and the tumor is preferably a non-solid tumor, more preferably multiple myeloma.

17. An antibody that binds to GPRC5D or its antigen-binding portion, comprising a heavy chain variable region whose amino acid sequence 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, 63, 64, 65, 66, or 68, and a light chain variable region whose amino acid sequence 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 or 69, Preferably, the antibody or its antigen-binding portion that binds to GPRC5D is (1) A heavy chain variable region whose amino acid sequence 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 Sequence ID No. 62, and a light chain variable region whose amino acid sequence 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 Sequence ID No. 67; (2) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 63, and light chain variable regions whose amino acid sequence 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 Sequence ID No. 67; (3) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 64, and light chain variable regions whose amino acid sequence 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 Sequence ID No. 67; (4) Heavy chain variable regions whose amino acid sequence 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 Sequence ID No. 65, and light chain variable regions whose amino acid sequence 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 Sequence ID No. 67; (5) A heavy chain variable region whose amino acid sequence 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: 66, and a light chain variable region whose amino acid sequence 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; or (6) Heavy chain variable regions whose amino acid sequence 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 light chain variable regions whose amino acid sequence 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 Includes, More preferably, the antibody or its antigen-binding portion that binds to GPRC5D is (1) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 62, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (2) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (3) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 64, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; (4) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 65, and a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 67; or (5) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 68, and light chain variable region containing the amino acid sequence shown in SEQ ID NO: 69 The antibody or its antigen-binding portion, including the antibody or its antigen-binding portion.

18. An antibody-drug conjugate comprising the antibody or its antigen-binding moiety according to claim 17.

19. An anti-GPRC5D chimeric antigen receptor comprising the antibody or its antigen-binding moiety as described in claim 17.

20. Engineered immune effector cells comprising the anti-GPRC5D chimeric antigen receptor according to claim 19.

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