Antigen-binding molecule that specifically binds to GPRC5D and CD3 and its medical use

Abstract

The present invention provides an antigen-binding molecule that specifically binds to GPRC5D and CD3, and a pharmaceutical use thereof. The antigen-binding molecule can be used to treat tumor-related diseases.

Description

[Technical field]

[0001] The present disclosure relates to the field of biotechnology, and more specifically, to antigen-binding molecules and uses thereof. [Background technology]

[0002] The statements herein are not necessarily intended to constitute prior art, but merely to provide background information relevant to the present disclosure.

[0003] Multiple myeloma (MM) is the second most common hematologic malignancy with a 5-year survival rate of approximately 53.9%. As the world population ages, the number of people affected is expected to increase.

[0004] One of the G proteins of GPRC5D binds to an orphan receptor, and the seven-transmembrane protein is specifically overexpressed in multiple myeloma patients and is associated with poor prognosis, but is barely expressed in most normal tissues. The expression level of GPR5CD is much higher in both newly diagnosed and relapsed / refractory MM patients than in healthy individuals. Furthermore, GPRC5D expression is not different between 1(q)gain and 13(q)del patients in various abnormal cell types. These associations between GPRC5D overexpression and cancer suggest that GPRC5D may serve as an excellent therapeutic target for cancer.

[0005] Currently, many therapeutic drugs are used clinically against MM, including immunomodulatory agents (IMIDs), protease inhibitors (PIs) and monoclonal antibody drugs. These drugs can certainly improve the clinical condition of MM patients, but these therapies each have their drawbacks, for example, IMIDs cause congenital defects, neurotoxicity and severe thrombosis, and PIs are associated with a high risk of peripheral neurotoxicity. Monoclonal antibodies against CD38 and SLAMF7 cause drug resistance due to antigen loss, as well as severe infections and anemia due to the reduction of red and white blood cells. At present, there is no therapy that can completely cure MM, and patients will eventually relapse. Therefore, the development of new targeted immunotherapy has important clinical implications. Summary of the Invention

[0006] The present disclosure provides antigen-binding molecules that specifically bind to GPRC5D and CD3, and antibodies that specifically bind to GPRC5D.

[0007] In one aspect, the present disclosure provides an antigen binding molecule that specifically binds to GPRC5D and CD3, comprising at least one antigen binding module that specifically binds to GPRC5D and at least one antigen binding module that specifically binds to CD3, wherein the antigen binding module that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, and the antigen binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL. In one aspect, the present disclosure provides an antigen-binding molecule that specifically binds to GPRC5D and CD3, comprising one or two antigen-binding modules that specifically bind to GPRC5D and one or two antigen-binding modules that specifically bind to CD3, wherein the antigen-binding module that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, and the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.

[0008] In one aspect, the present disclosure provides an antigen-binding molecule that specifically binds to GPRC5D and CD3, comprising one antigen-binding module that specifically binds to GPRC5D and one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, and the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.

[0009] In one aspect, the present disclosure provides an antigen-binding molecule that specifically binds to GPRC5D and CD3, comprising two antigen-binding modules that specifically bind to GPRC5D and two antigen-binding modules that specifically bind to CD3, wherein the antigen-binding module that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, and the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.

[0010] In an embodiment of an antigen-binding molecule that specifically binds to GPRC5D and CD3 provided in the present disclosure, the antigen-binding module that specifically binds to GPRC5D may be a Fab, which includes a heavy chain variable region GPRC5D-VH, a heavy chain constant region CH1, a light chain variable region GPRC5D-VL, and a light chain constant region CL.

[0011] In an embodiment of an antigen-binding molecule that specifically binds to GPRC5D and CD3 provided in the present disclosure, the antigen-binding module that specifically binds to CD3 may be a substituted Fab, which includes a heavy chain variable region CD3-VH, an Obscurin chain, a light chain variable region CD3-VL, and a Titin chain.

[0012] In an embodiment of the antigen-binding molecule that specifically binds to GPRC5D and CD3 provided in the present disclosure, the antigen-binding module that specifically binds to CD3 may be an scFv and includes a heavy chain variable region CD3-VH and a light chain variable region CD3-VL.

[0013] In some embodiments, the antigen-binding molecule comprises: (i) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, 18, 135, 136 or 138, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, 10 or 133, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, 12, 129, 131 or 132, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or (iii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 140, 22 or 139, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or (iv) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 144, 30, 141, 142 or 143, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; Antigen-binding molecules.

[0014] In some embodiments, GPRC5D-HCDR1, GPRC5D-HCDR2, GPRC5D-HCDR3, GPRC5D-LCDR1, GPRC5D-LCDR2 and GPRC5D-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

[0015] In some embodiments, the antigen-binding molecule comprises: (i) the GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 18, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 138, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 129, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 133, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 133, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 133, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 129, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or (iii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 139, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or (iv) the GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 144, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; Antigen-binding molecules.

[0016] In some embodiments, the antigen-binding molecule of any one of the above, (i) the GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; Antigen-binding molecules.

[0017] In some embodiments, the antigen-binding molecule of any one of the above, The GPRC5D-VH comprises GPRC5D-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 9, GPRC5D-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 134, and GPRC5D-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 11, and the GPRC5D-VL comprises GPRC5D-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 130, GPRC5D-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 13, and GPRC5D-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 14. Antigen-binding molecules.

[0018] In an embodiment of the antigen binding molecule of the present disclosure that specifically binds to GPRC5D and CD3, (i) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 15, GPRC5D-HCDR2 shown in SEQ ID NO: 16, and GPRC5D-HCDR3 shown in SEQ ID NO: 17; and The GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 137, 18, 135, 136 or 138, GPRC5D-LCDR2 shown in SEQ ID NO: 19, and GPRC5D-LCDR3 shown in SEQ ID NO: 20, or (ii) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 9, GPRC5D-HCDR2 shown in SEQ ID NO: 134, 10 or 133, and GPRC5D-HCDR3 shown in SEQ ID NO: 11; and The GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 130, 12, 129, 131 or 132, GPRC5D-LCDR2 shown in SEQ ID NO: 13, and GPRC5D-LCDR3 shown in SEQ ID NO: 14, or (iii) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 21, GPRC5D-HCDR2 shown in SEQ ID NO: 140, 22 or 139, and GPRC5D-HCDR3 shown in SEQ ID NO: 23; and The GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 24, GPRC5D-LCDR2 shown in SEQ ID NO: 25, and GPRC5D-LCDR3 shown in SEQ ID NO: 26, or (iv) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 27, GPRC5D-HCDR2 shown in SEQ ID NO: 28, and GPRC5D-HCDR3 shown in SEQ ID NO: 29; and The GPRC5D-VL includes GPRC5D-LCDR1 represented by SEQ ID NO: 144, 30, 141, 142 or 143, GPRC5D-LCDR2 represented by SEQ ID NO: 31, and GPRC5D-LCDR3 represented by SEQ ID NO: 32, Preferably, (i) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 15, GPRC5D-HCDR2 shown in SEQ ID NO: 16, and GPRC5D-HCDR3 shown in SEQ ID NO: 17; and The GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 137, GPRC5D-LCDR2 shown in SEQ ID NO: 19, and GPRC5D-LCDR3 shown in SEQ ID NO: 20, or (ii) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 9, GPRC5D-HCDR2 shown in SEQ ID NO: 134, and GPRC5D-HCDR3 shown in SEQ ID NO: 11; and The above GPRC5D-VL comprises GPRC5D-LCDR1 shown in SEQ ID NO:130, GPRC5D-LCDR2 shown in SEQ ID NO:13, and GPRC5D-LCDR3 shown in SEQ ID NO:14.

[0019] In some embodiments, the antigen-binding molecule of any one of the above, The GPRC5D-VH comprises GPRC5D-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 15, GPRC5D-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 16, and GPRC5D-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 17, and the GPRC5D-VL comprises GPRC5D-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 137, GPRC5D-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 19, and GPRC5D-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 20. Antigen-binding molecules.

[0020] In some embodiments, GPRC5D-HCDR1, GPRC5D-HCDR2, GPRC5D-HCDR3, GPRC5D-LCDR1, GPRC5D-LCDR2 and GPRC5D-LCDR3 are defined according to the Kabat numbering convention.

[0021] In some embodiments, the antigen-binding molecule of any one of the above, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, 3, 42 or 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, 1, 33 or 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, 5, 51, 52, 53 or 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56, 6 or 57; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, 7, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 68, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75, 8, 69, 70, 71, 72, 73 or 74; Antigen-binding molecules.

[0022] In some embodiments, the antigen-binding molecule of any one of the above, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 3, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 4; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 1, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 2; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 57; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 57; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:5, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:6; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 61, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 62, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 64, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 65, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 7, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 8; Antigen-binding molecules.

[0023] In some embodiments, the antigen-binding molecule of any one of the above, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; Antigen-binding molecules.

[0024] In some embodiments, the antigen-binding molecule of any one of the above, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; Antigen-binding molecules.

[0025] In some embodiments, the antigen-binding molecule of any one of the above, The amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 35, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 39. Antigen-binding molecules.

[0026] In some embodiments, the antigen-binding molecule of any one of the above, The amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 44, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 49. Antigen-binding molecules.

[0027] In an embodiment of the antigen-binding molecule of the present disclosure that specifically binds to GPRC5D and CD3, the antigen-binding module that specifically binds to GPRC5D is (i) GPRC5D-VH represented by SEQ ID NO: 44, 3, 42 or 43, and GPRC5D-VL represented by SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50; or (ii) GPRC5D-VH represented by SEQ ID NO: 35, 1, 33 or 34, and GPRC5D-VL represented by SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41; or (iii) GPRC5D-VH represented by SEQ ID NO: 55, 5, 51, 52, 53 or 54, and GPRC5D-VL represented by SEQ ID NO: 56, 6 or 57; or (iv) GPRC5D-VH represented by SEQ ID NO: 67, 7, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 68, and GPRC5D-VL represented by SEQ ID NO: 75, 8, 69, 70, 71, 72, 73 or 74; Including, Preferably, (i) GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 49, or GPRC5D-VH shown in SEQ ID NO: 43 and GPRC5D-VL shown in SEQ ID NO: 46, or GPRC5D-VH shown in SEQ ID NO: 43 and GPRC5D-VL shown in SEQ ID NO: 49, or GPRC5D-VH as shown in SEQ ID NO: 43 and GPRC5D-VL as shown in SEQ ID NO: 50, or GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 46, or GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 50, or GPRC5D-VH shown in SEQ ID NO: 3 and GPRC5D-VL shown in SEQ ID NO: 4, or (ii) GPRC5D-VH shown in SEQ ID NO: 35 and GPRC5D-VL shown in SEQ ID NO: 39, or GPRC5D-VH shown in SEQ ID NO: 34 and GPRC5D-VL shown in SEQ ID NO: 36, or GPRC5D-VH shown in SEQ ID NO: 34 and GPRC5D-VL shown in SEQ ID NO: 38, or GPRC5D-VH shown in SEQ ID NO: 34 and GPRC5D-VL shown in SEQ ID NO: 39, or GPRC5D-VH shown in SEQ ID NO: 35 and GPRC5D-VL shown in SEQ ID NO: 36, or GPRC5D-VH shown in SEQ ID NO: 35 and GPRC5D-VL shown in SEQ ID NO: 38, or GPRC5D-VH shown in SEQ ID NO: 1 and GPRC5D-VL shown in SEQ ID NO: 2, or (iii) GPRC5D-VH shown in SEQ ID NO: 55 and GPRC5D-VL shown in SEQ ID NO: 56, or GPRC5D-VH as shown in SEQ ID NO: 54 and GPRC5D-VL as shown in SEQ ID NO: 56, or GPRC5D-VH shown in SEQ ID NO: 54 and GPRC5D-VL shown in SEQ ID NO: 57, or GPRC5D-VH as shown in SEQ ID NO: 55 and GPRC5D-VL as shown in SEQ ID NO: 57, or GPRC5D-VH shown in SEQ ID NO: 5 and GPRC5D-VL shown in SEQ ID NO: 6, or (iv) GPRC5D-VH shown in SEQ ID NO: 67 and GPRC5D-VL shown in SEQ ID NO: 75, or GPRC5D-VH as shown in SEQ ID NO: 61 and GPRC5D-VL as shown in SEQ ID NO: 75, or GPRC5D-VH as shown in SEQ ID NO: 62 and GPRC5D-VL as shown in SEQ ID NO: 75, or GPRC5D-VH shown in SEQ ID NO: 64 and GPRC5D-VL shown in SEQ ID NO: 75, or GPRC5D-VH shown in SEQ ID NO: 65 and GPRC5D-VL shown in SEQ ID NO: 75, or GPRC5D-VH shown in SEQ ID NO: 7 and GPRC5D-VL shown in SEQ ID NO: 8, or More preferably, the antigen-binding module that specifically binds to GPRC5D is (i) GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 49, or (ii) GPRC5D-VH shown in SEQ ID NO: 35, and GPRC5D-VL shown in SEQ ID NO: 39; Includes.

[0028] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule further comprises an Fc region, the Fc region being preferably an IgG Fc region, more preferably an IgG1 Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions compared to a wild-type Fc region, the amino acid substitutions being capable of reducing binding to an Fc receptor, in particular binding to an Fcγ receptor. In some embodiments, the Fc region comprises one or more amino acid substitutions being capable of reducing binding of the Fc region to an Fcγ receptor. In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 97.

[0029] In some embodiments, the antigen-binding molecule is any one of the above, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2) capable of associating with each other, and the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce Fc region homodimerization. In some embodiments, the Fc1 and Fc2 each independently have one or more amino acid substitutions by knob-in-hole technology. In some embodiments, the Fc1 has a knob structure by knob-in-hole technology and the Fc2 has a hole structure by knob-in-hole technology, or the Fc1 has a hole structure by knob-in-hole technology and the Fc2 has a knob structure by knob-in-hole technology, preferably the Fc1 has a knob structure by knob-in-hole technology and the Fc2 has a hole structure by knob-in-hole technology. In some embodiments, the Fc1 has an amino acid at position 366 that is W, and the Fc2 has an amino acid at position 366 that is S, an amino acid at position 368 that is A, and an amino acid at position 407 that is V, numbered according to the EU index, or vice versa. In some embodiments, the Fc1 has an amino acid at position 354 that is C, and the Fc2 has an amino acid at position 349 that is C, numbered according to the EU index, or vice versa. In some embodiments, the Fc1 has an amino acid at position 354 that is C, an amino acid at position 366 that is W, and the Fc2 has an amino acid at position 349 that is C, an amino acid at position 366 that is S, an amino acid at position 368 that is A, and an amino acid at position 407 that is V, numbered according to the EU index, or vice versa. In some embodiments, the Fc1 has the amino acid sequence of SEQ ID NO:92, and the Fc2 has the amino acid sequence of SEQ ID NO:93.

[0030] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises one or two antigen-binding modules that specifically bind to GPRC5D and one or two antigen-binding modules that specifically bind to CD3. In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises one antigen-binding module that specifically binds to GPRC5D and one antigen-binding module that specifically binds to CD3. In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding molecule comprises two antigen-binding modules that specifically bind to GPRC5D and two antigen-binding modules that specifically bind to CD3.

[0031] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding module that specifically binds to GPRC5D is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab or scFv. In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding module that specifically binds to GPRC5D is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab. In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding module that specifically binds to GPRC5D is a Fab, and the antigen-binding module that specifically binds to CD3 is an scFv. In some embodiments, the substituted Fab has the original CH1 and CL of the Fab replaced with the Obscurin chain and the Titin chain, respectively, or the original CH1 and CL of the Fab replaced with the Titin chain and the Obscurin chain, respectively.

[0032] In some embodiments, the antigen-binding molecule according to any one of the above, wherein the antigen-binding module that specifically binds to GPRC5D or the antigen-binding module that specifically binds to CD3 comprises a Titin chain and an Obscurin chain capable of forming a dimer. In some embodiments, the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145 to 163, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 164 to 204. In some embodiments, the Titin chain comprises the amino acid sequence of SEQ ID NO: 163 or 161, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 198 or 199. In some embodiments, the Titin chain comprises the amino acid sequence of SEQ ID NO: 163, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the Titin chain comprises the amino acid sequence of SEQ ID NO: 161, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 199.

[0033] In some embodiments, the antigen-binding molecule comprises one antigen-binding module that specifically binds to GPRC5D and one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to GPRC5D is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab that comprises a Titin chain and an Obscurin chain capable of forming a dimer, preferably, the original CH1 and CL of the Fab are replaced with the Obscurin chain and the Titin chain, respectively, or the original CH1 and CL of the Fab are replaced with the Titin chain and the Obscurin chain, respectively.

[0034] In some embodiments, at least one of the antigen-binding modules that specifically binds to GPRC5D and at least one of the antigen-binding modules that specifically binds to CD3 are linked to the N-terminus of the Fc1 or Fc2 subunit directly or via a linker at the C-terminus of the CH1 or Obscurin chain, and are associated as a heterodimer by disulfide bonds at the N-terminus of the Fc1 and Fc2 subunits.

[0035] In some embodiments, the antigen-binding molecule comprises one first chain having the structure shown in formula (a), one second chain having the structure shown in formula (b), one third chain having the structure shown in formula (c), and one fourth chain having the structure shown in formula (d); (a) [GPRC5D-VH]-[CH1]-[Fc1], (b) [GPRC5D-VL]-CL, (c) [CD3-VH]-[Obscurin chain]-[Fc2], (d) [CD3-VL]-[Titin chain], The structures represented by formulae (a), (b), (c) and (d) are arranged from the N-terminus to the C-terminus.

[0036] In some embodiments, the antigen binding molecule is any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 35, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 39, the Obscurin chain in the third chain comprises the amino acid sequence of SEQ ID NO: 198, and the Titin chain in the fourth chain comprises the amino acid sequence of SEQ ID NO: 163.

[0037] In some embodiments, the antigen-binding molecule has a first chain comprising the amino acid sequence of SEQ ID NO: 118, a second chain comprising the amino acid sequence of SEQ ID NO: 102, a third chain comprising the amino acid sequence of SEQ ID NO: 86, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 87.

[0038] In some embodiments, the antigen-binding molecule has a first chain having a structure represented by formula (e), a second chain having a structure represented by formula (d), a third chain having a structure represented by formula (f), and a fourth chain having a structure represented by formula (b); (e) [CD3-VH]-[Obscurin chain]-[Fc1], (d) [CD3-VL]-[Titin chain], (f) [GPRC5D-VH]-[CH1]-[Fc2]; (b) [GPRC5D-VL]-CL, The structures represented by formulae (e), (d), (f) and (b) are arranged from the N-terminus to the C-terminus.

[0039] In some embodiments, the antigen binding molecule described above, wherein the Obscurin chain in the first chain comprises the amino acid sequence of SEQ ID NO: 198, the Titin chain in the second chain comprises the amino acid sequence of SEQ ID NO: 163, the GPRC5D-VH in the third chain comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL in the fourth chain comprises the amino acid sequence of SEQ ID NO: 49.

[0040] In some embodiments, the antigen-binding molecule has a first chain comprising the amino acid sequence of SEQ ID NO: 89, a second chain comprising the amino acid sequence of SEQ ID NO: 87, a third chain comprising the amino acid sequence of SEQ ID NO: 121, and a fourth chain comprising the amino acid sequence of SEQ ID NO: 106.

[0041] In some embodiments, the antigen-binding molecule comprises one first chain having the structure shown in formula (a), one second chain having the structure shown in formula (b), one third chain having the structure shown in formula (g), and one fourth chain having the structure shown in formula (h); (a) [GPRC5D-VH]-[CH1]-[Fc1], (b) [GPRC5D-VL]-CL, (g) [CD3-VH]-linker1-[Obscurin chain]-[Fc2], (h) [CD3-VL]-linker2-[Titin chain], The structures represented by formulae (a), (b), (g) and (h) are arranged from the N-terminus to the C-terminus, and the above linker 1 and linker 2 are the same or different peptide linkers.

[0042] In some embodiments, the antigen-binding molecule described above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 35, 44, 55, or 67, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 39, 49, 56, or 75, the Obscurin chain in the third chain comprises the amino acid sequence of SEQ ID NO: 199, and the Titin chain in the fourth chain comprises the amino acid sequence of SEQ ID NO: 161. In some embodiments, linker 1 and / or linker 2 comprise the amino acid sequence of (GGGGS)n, wherein n is 1 to 5, preferably n is 1, 2, or 3, and most preferably n is 1.

[0043] In some embodiments, the antigen-binding molecule of any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 35, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 39, the Obscurin chain in the third chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the fourth chain comprises the amino acid sequence of SEQ ID NO: 161, and the amino acid sequences of linker 1 and linker 2 are set forth in SEQ ID NO: 90. In some embodiments, the antigen-binding molecule of any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 44, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 49, the Obscurin chain in the third chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the fourth chain comprises the amino acid sequence of SEQ ID NO: 161, and the amino acid sequences of linker 1 and linker 2 are set forth in SEQ ID NO: 90. In some embodiments, the antigen-binding molecule of any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 55, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 56, the Obscurin chain in the third chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the fourth chain comprises the amino acid sequence of SEQ ID NO: 161, and the amino acid sequences of linker 1 and linker 2 are set forth in SEQ ID NO: 90. In some embodiments, the antigen-binding molecule of any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 67, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 75, the Obscurin chain in the third chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the fourth chain comprises the amino acid sequence of SEQ ID NO: 161, and the amino acid sequences of linker 1 and linker 2 are set forth in SEQ ID NO: 90.

[0044] In some embodiments, the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 118, one second chain comprising the amino acid sequence of SEQ ID NO: 102, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 120, one second chain comprising the amino acid sequence of SEQ ID NO: 106, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 110, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 124, one second chain comprising the amino acid sequence of SEQ ID NO: 117, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85.

[0045] In some embodiments, the antigen-binding molecule comprises one first chain having the structure represented by formula (i), one second chain having the structure represented by formula (j), one third chain having the structure represented by formula (f), and one fourth chain having the structure represented by formula (b); (i) [CD3-VH]-linker3-[Obscurin chain]-[Fc1], (j) [CD3-VL]-linker4-[Titin chain], (f) [GPRC5D-VH]-[CH1]-[Fc2]; (b) [GPRC5D-VL]-CL, The structures represented by formulae (i), (j), (f) and (b) are arranged from the N-terminus to the C-terminus, and the linker 3 and the linker 4 are the same or different peptide linkers.

[0046] In some embodiments, the antigen-binding molecule described above, wherein the Obscurin chain in the first chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the second chain comprises the amino acid sequence of SEQ ID NO: 161, the GPRC5D-VH in the third chain comprises the amino acid sequence of SEQ ID NO: 35, 44, 55, 61, 62, 64, 65, or 67, and the GPRC5D-VL in the fourth chain comprises the amino acid sequence of SEQ ID NO: 39, 49, 56, or 75. In some embodiments, linker 3 and / or linker 4 comprise the amino acid sequence of (GGGGS)n, wherein n is 1 to 5, preferably n is 1, 2, or 3, and most preferably n is 1.

[0047] In some embodiments, the antigen-binding molecule of any one of the above, wherein the Obscurin chain in the first chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the second chain comprises the amino acid sequence of SEQ ID NO: 161, the GPRC5D-VH in the third chain comprises the amino acid sequence of SEQ ID NO: 35, the GPRC5D-VL in the fourth chain comprises the amino acid sequence of SEQ ID NO: 39, and the amino acid sequences of linker 3 and linker 4 are set forth in SEQ ID NO: 90. In some embodiments, the antigen-binding molecule of any one of the above, wherein the Obscurin chain in the first chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the second chain comprises the amino acid sequence of SEQ ID NO: 161, the GPRC5D-VH in the third chain comprises the amino acid sequence of SEQ ID NO: 44, the GPRC5D-VL in the fourth chain comprises the amino acid sequence of SEQ ID NO: 49, and the amino acid sequences of linker 3 and linker 4 are set forth in SEQ ID NO: 90. In some embodiments, the antigen-binding molecule is any one of the above, wherein the Obscurin chain in the first chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the second chain comprises the amino acid sequence of SEQ ID NO: 161, the GPRC5D-VH in the third chain comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL in the fourth chain comprises the amino acid sequence of SEQ ID NO: 56, and the amino acid sequences of linker 3 and linker 4 are set forth in SEQ ID NO: 90. In some embodiments, the antigen binding molecule is any one of the above, wherein the Obscurin chain in the first chain comprises the amino acid sequence of SEQ ID NO: 199, the Titin chain in the second chain comprises the amino acid sequence of SEQ ID NO: 161, the GPRC5D-VH in the third chain comprises the amino acid sequence of SEQ ID NO: 61, 62, 64, 65 or 67, the GPRC5D-VL in the fourth chain comprises the amino acid sequence of SEQ ID NO: 75, and the amino acid sequences of linker 3 and linker 4 are set forth in SEQ ID NO: 90.

[0048] In some embodiments, the antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 116, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 117; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 121, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 106; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 123, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 110; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 119, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 102; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 115, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 117; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 112, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 117; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 113, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 117; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 114, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 117.

[0049] The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 227, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 223.

[0050] In some embodiments, the antigen-binding molecule comprises two antigen-binding modules that specifically bind to GPRC5D and two antigen-binding modules that specifically bind to CD3, wherein the antigen-binding module that specifically binds to GPRC5D is a Fab and the antigen-binding module that specifically binds to CD3 is an scFv.

[0051] In some embodiments, the antigen-binding molecule comprises two first chains having the structure shown in formula (k) and two second chains having the structure shown in formula (b), (k) [GPRC5D-VH]-[CH1]-[CD3-VH]-linker5-[CD3-VL]-linker6-[Fc3]; (b) [GPRC5D-VL]-CL, The structures represented by formulae (k) and (b) are arranged from the N-terminus to the C-terminus, and the above linkers 5 and 6 are the same or different peptide linkers.

[0052] In some embodiments, the antigen-binding portion as described above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 35, 34, 44, 43, 55, or 54, and the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 39, 36, 38, 49, 46, 50, 56, or 57. In some embodiments, Linker 5 and / or Linker 6 comprise the amino acid sequence of (GGGGS)n, wherein n is 1 to 5, preferably n is 1, 2, or 3, and most preferably n is 1 or 3.

[0053] In some embodiments, the antigen-binding molecule described in any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 35 or 34, and the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 39, 36 or 38, the amino acid sequence of the linker 5 is represented by SEQ ID NO: 94, and the amino acid sequence of the linker 6 is represented by SEQ ID NO: 95. In some embodiments, the antigen-binding molecule described in any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 44 or 43, and the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 49, 46 or 50, the amino acid sequence of the linker 5 is represented by SEQ ID NO: 94, and the amino acid sequence of the linker 6 is represented by SEQ ID NO: 95. In some embodiments, the antigen-binding molecule is any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 55 or 54, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 56 or 57, the amino acid sequence of linker 5 is represented by SEQ ID NO: 94, and the amino acid sequence of linker 6 is represented by SEQ ID NO: 95.

[0054] In some embodiments, the antigen-binding molecule is any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 35, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 39, the amino acid sequence of linker 5 is represented by SEQ ID NO: 94, and the amino acid sequence of linker 6 is represented by SEQ ID NO: 95.

[0055] In some embodiments, the antigen-binding molecule is any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 44, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 49, the amino acid sequence of linker 5 is represented by SEQ ID NO: 94, and the amino acid sequence of linker 6 is represented by SEQ ID NO: 95.

[0056] In some embodiments, the antigen-binding molecule is any one of the above, wherein the GPRC5D-VH in the first chain comprises the amino acid sequence of SEQ ID NO: 55, the GPRC5D-VL in the second chain comprises the amino acid sequence of SEQ ID NO: 56, the amino acid sequence of linker 5 is represented by SEQ ID NO: 94, and the amino acid sequence of linker 6 is represented by SEQ ID NO: 95.

[0057] In some embodiments, the antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 98 and two second chains comprising the amino acid sequence of SEQ ID NO: 100; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 98 and two second chains comprising the amino acid sequence of SEQ ID NO: 101; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 98 and two second chains comprising the amino acid sequence of SEQ ID NO: 102; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 99 and two second chains comprising the amino acid sequence of SEQ ID NO: 100; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 99 and two second chains comprising the amino acid sequence of SEQ ID NO: 101; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 99 and two second chains comprising the amino acid sequence of SEQ ID NO: 102; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 103 and two second chains comprising the amino acid sequence of SEQ ID NO: 105; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 103 and two second chains comprising the amino acid sequence of SEQ ID NO: 106; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 103 and two second chains comprising the amino acid sequence of SEQ ID NO: 107; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 104 and two second chains comprising the amino acid sequence of SEQ ID NO: 105; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 104 and two second chains comprising the amino acid sequence of SEQ ID NO: 106; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 104 and two second chains comprising the amino acid sequence of SEQ ID NO: 107; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 108 and two second chains comprising the amino acid sequence of SEQ ID NO: 110; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 109 and two second chains comprising the amino acid sequence of SEQ ID NO: 110; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 108 and two second chains comprising the amino acid sequence of SEQ ID NO: 111; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 109 and two second chains comprising the amino acid sequence of SEQ ID NO: 111; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 224 and two second chains comprising the amino acid sequence of SEQ ID NO: 217; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 225 and two second chains comprising the amino acid sequence of SEQ ID NO: 219; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO:226 and two second chains comprising the amino acid sequence of SEQ ID NO:221.

[0058] In another aspect, the present disclosure provides an isolated antibody capable of specifically binding to GPRC5D, said isolated antibody comprising a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein: (i) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, 18, 135, 136 or 138, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, 10 or 133, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, 12, 129, 131 or 132, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or (iii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 140, 22 or 139, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or (iv) The above GPRC5D-VH has GPRC5D-HCDR1 having the amino acid sequence of SEQ ID NO: 27, GPRC5D-HCDR2 having the amino acid sequence of SEQ ID NO: 28, and GPRC5D-HCDR3 having the amino acid sequence of SEQ ID NO: 29, and the above GPRC5D-VL has GPRC5D-LCDR1 having the amino acid sequence of SEQ ID NO: 144, 30, 141, 142 or 143, GPRC5D-LCDR2 having the amino acid sequence of SEQ ID NO: 31, and GPRC5D-LCDR3 having the amino acid sequence of SEQ ID NO: 32.

[0059] In some embodiments, GPRC5D-HCDR1, GPRC5D-HCDR2, GPRC5D-HCDR3, GPRC5D-LCDR1, GPRC5D-LCDR2 and GPRC5D-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

[0060] In some embodiments, the isolated antibody is an antibody comprising: (i) the GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 18, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 138, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 129, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 133, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 133, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 133, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 129, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, or (iii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or The GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 139, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or (iv) the GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 144, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; Isolated antibodies.

[0061] In some embodiments, the isolated antibody is an antibody comprising: (i) the GPRC5D-VH has GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20, or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; Isolated antibodies.

[0062] In some embodiments, an isolated antibody capable of specifically binding to GPRC5D is provided, the antibody comprising a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein: (i) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 15, GPRC5D-HCDR2 shown in SEQ ID NO: 16, and GPRC5D-HCDR3 shown in SEQ ID NO: 17; and The GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 137, 18, 135, 136 or 138, GPRC5D-LCDR2 shown in SEQ ID NO: 19, and GPRC5D-LCDR3 shown in SEQ ID NO: 20, or (ii) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 9, GPRC5D-HCDR2 shown in SEQ ID NO: 134, 10 or 133, and GPRC5D-HCDR3 shown in SEQ ID NO: 11; and The GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 130, 12, 129, 131 or 132, GPRC5D-LCDR2 shown in SEQ ID NO: 13, and GPRC5D-LCDR3 shown in SEQ ID NO: 14, or (iii) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 21, GPRC5D-HCDR2 shown in SEQ ID NO: 140, 22 or 139, and GPRC5D-HCDR3 shown in SEQ ID NO: 23; and The GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 24, GPRC5D-LCDR2 shown in SEQ ID NO: 25, and GPRC5D-LCDR3 shown in SEQ ID NO: 26, or (iv) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 27, GPRC5D-HCDR2 shown in SEQ ID NO: 28, and GPRC5D-HCDR3 shown in SEQ ID NO: 29; and The GPRC5D-VL includes GPRC5D-LCDR1 represented by SEQ ID NO: 144, 30, 141, 142 or 143, GPRC5D-LCDR2 represented by SEQ ID NO: 31, and GPRC5D-LCDR3 represented by SEQ ID NO: 32, Preferably, (i) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 15, GPRC5D-HCDR2 shown in SEQ ID NO: 16, and GPRC5D-HCDR3 shown in SEQ ID NO: 17; And the GPRC5D-VL includes GPRC5D-LCDR1 shown in SEQ ID NO: 137, GPRC5D-LCDR2 shown in SEQ ID NO: 19, and GPRC5D-LCDR3 shown in SEQ ID NO: 20, or (ii) the GPRC5D-VH comprises GPRC5D-HCDR1 shown in SEQ ID NO: 9, GPRC5D-HCDR2 shown in SEQ ID NO: 134, and GPRC5D-HCDR3 shown in SEQ ID NO: 11; and the GPRC5D-VL comprises GPRC5D-LCDR1 shown in SEQ ID NO: 130, GPRC5D-LCDR2 shown in SEQ ID NO: 13, and GPRC5D-LCDR3 shown in SEQ ID NO: 14; Isolated antibodies.

[0063] In some embodiments, the isolated antibody is an antibody comprising: The GPRC5D-VH comprises GPRC5D-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 9, GPRC5D-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 134, and GPRC5D-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 11, and the GPRC5D-VL comprises GPRC5D-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 130, GPRC5D-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 13, and GPRC5D-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 14. In some embodiments, the isolated antibody is an antibody as described above, The GPRC5D-VH comprises GPRC5D-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 15, GPRC5D-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 16, and GPRC5D-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 17, and the GPRC5D-VL comprises GPRC5D-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 137, GPRC5D-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 19, and GPRC5D-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 20. Isolated antibodies.

[0064] In some embodiments, GPRC5D-HCDR1, GPRC5D-HCDR2, GPRC5D-HCDR3, GPRC5D-LCDR1, GPRC5D-LCDR2 and GPRC5D-LCDR3 are defined according to the Kabat numbering convention.

[0065] In some embodiments, the isolated antibody is an antibody comprising: (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, 3, 42 or 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, 1, 33 or 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, 5, 51, 52, 53 or 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56, 6 or 57; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, 7, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 68, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75, 8, 69, 70, 71, 72, 73 or 74; Isolated antibodies.

[0066] In some embodiments, the isolated antibody is an antibody comprising: (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 3, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 4; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 1, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 2; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 57; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 57; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:5, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:6; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 61, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 62, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 64, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 65, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 7, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 8; Isolated antibodies.

[0067] In some embodiments, the isolated antibody is an antibody comprising: (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; Isolated antibodies.

[0068] In some embodiments, the isolated antibody is an antibody comprising: (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; Isolated antibodies.

[0069] In some embodiments, the isolated antibody is an antibody comprising: The amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 35, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 39. Isolated antibodies.

[0070] In some embodiments, the isolated antibody is an antibody comprising: The amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 44, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 49. Isolated antibodies.

[0071] In some embodiments, the isolated antibody comprises: (i) GPRC5D-VH represented by SEQ ID NO: 44, 3, 42 or 43, and GPRC5D-VL represented by SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50; or (ii) GPRC5D-VH represented by SEQ ID NO: 35, 1, 33 or 34, and GPRC5D-VL represented by SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41; or (iii) GPRC5D-VH represented by SEQ ID NO: 55, 5, 51, 52, 53 or 54, and GPRC5D-VL represented by SEQ ID NO: 56, 6 or 57; or (iv) GPRC5D-VH represented by SEQ ID NO: 67, 7, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 68, and GPRC5D-VL represented by SEQ ID NO: 75, 8, 69, 70, 71, 72, 73 or 74; Including, Preferably, the antibody comprises (i) GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 49, or GPRC5D-VH shown in SEQ ID NO: 43 and GPRC5D-VL shown in SEQ ID NO: 46, or GPRC5D-VH shown in SEQ ID NO: 43 and GPRC5D-VL shown in SEQ ID NO: 49, or GPRC5D-VH as shown in SEQ ID NO: 43 and GPRC5D-VL as shown in SEQ ID NO: 50, or GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 46, or GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 50, or GPRC5D-VH shown in SEQ ID NO: 3 and GPRC5D-VL shown in SEQ ID NO: 4, or (ii) GPRC5D-VH shown in SEQ ID NO: 35 and GPRC5D-VL shown in SEQ ID NO: 39, or GPRC5D-VH shown in SEQ ID NO: 34 and GPRC5D-VL shown in SEQ ID NO: 36, or GPRC5D-VH shown in SEQ ID NO: 34 and GPRC5D-VL shown in SEQ ID NO: 38, or GPRC5D-VH shown in SEQ ID NO: 34 and GPRC5D-VL shown in SEQ ID NO: 39, or GPRC5D-VH shown in SEQ ID NO: 35 and GPRC5D-VL shown in SEQ ID NO: 36, or GPRC5D-VH shown in SEQ ID NO: 35 and GPRC5D-VL shown in SEQ ID NO: 38, or GPRC5D-VH shown in SEQ ID NO: 1 and GPRC5D-VL shown in SEQ ID NO: 2, or (iii) GPRC5D-VH shown in SEQ ID NO: 55 and GPRC5D-VL shown in SEQ ID NO: 56, or GPRC5D-VH as shown in SEQ ID NO: 54 and GPRC5D-VL as shown in SEQ ID NO: 56, or GPRC5D-VH shown in SEQ ID NO: 54 and GPRC5D-VL shown in SEQ ID NO: 57, or GPRC5D-VH as shown in SEQ ID NO: 55 and GPRC5D-VL as shown in SEQ ID NO: 57, or GPRC5D-VH shown in SEQ ID NO: 5 and GPRC5D-VL shown in SEQ ID NO: 6, or (iv) GPRC5D-VH shown in SEQ ID NO: 67 and GPRC5D-VL shown in SEQ ID NO: 75, or GPRC5D-VH as shown in SEQ ID NO: 61 and GPRC5D-VL as shown in SEQ ID NO: 75, or GPRC5D-VH as shown in SEQ ID NO: 62 and GPRC5D-VL as shown in SEQ ID NO: 75, or GPRC5D-VH shown in SEQ ID NO: 64 and GPRC5D-VL shown in SEQ ID NO: 75, or GPRC5D-VH shown in SEQ ID NO: 65 and GPRC5D-VL shown in SEQ ID NO: 75, or Contains GPRC5D-VH shown in SEQ ID NO: 7 and GPRC5D-VL shown in SEQ ID NO: 8, or More preferably, the antibody (i) GPRC5D-VH shown in SEQ ID NO: 44 and GPRC5D-VL shown in SEQ ID NO: 49, or (ii) GPRC5D-VH of SEQ ID NO: 35, and GPRC5D-VL of SEQ ID NO: 39; Includes.

[0072] In some embodiments, the antigen binding molecule of any one of the above, or the isolated antibody of any one of the above, is a bispecific antibody.

[0073] In some embodiments, the bispecific antibody specifically binds to GPRC5D and CD3.

[0074] In some embodiments, the antigen-binding molecule or antibody described in any one of the above is an antigen-binding molecule or an isolated antibody described in any one of the above, wherein the antigen-binding molecule or antibody does not bind to an E2 peptide, an E3 peptide, or an E4 peptide, wherein the E2 peptide comprises the amino acid sequence of SEQ ID NO: 206, the E3 peptide comprises the amino acid sequence of SEQ ID NO: 207, and the E4 peptide comprises the amino acid sequence of SEQ ID NO: 208.

[0075] In some embodiments, the antigen binding molecule described in any one of the above or the isolated antibody described in any one of the above binds to the amino terminal region of human GPRC5D, wherein the amino terminal region of human GPRC5D comprises the amino acid sequence of SEQ ID NO: 205.

[0076] In some embodiments, the antigen-binding molecule or antibody described in any one of the above is an antigen-binding molecule or an isolated antibody described in any one of the above, wherein the antigen-binding molecule or antibody does not bind to E2 peptide, E3 peptide, or E4 peptide, and binds to the amino-terminal region of human GPRC5D, wherein the E2 peptide comprises the amino acid sequence of SEQ ID NO: 206, the E3 peptide comprises the amino acid sequence of SEQ ID NO: 207, the E4 peptide comprises the amino acid sequence of SEQ ID NO: 208, and the amino-terminal region comprises the amino acid sequence of SEQ ID NO: 205.

[0077] In some embodiments, the present invention is an antigen-binding molecule described in any one of the above, or an isolated antibody described in any one of the above, wherein the antigen-binding molecule or antibody does not bind to an E2 peptide, an E3 peptide, or an E4 peptide, and binds to the amino-terminal region of human GPRC5D, wherein the amino acid sequence of the E2 peptide is represented by SEQ ID NO: 206, the amino acid sequence of the E3 peptide is represented by SEQ ID NO: 207, the amino acid sequence of the E4 peptide is represented by SEQ ID NO: 208, and the amino acid sequence of the amino-terminal region is represented by SEQ ID NO: 205.

[0078] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the antigen-binding molecules described above or any one of the isolated antibodies described above, and one or more pharma- ceutically acceptable vectors, diluents, buffers, or excipients. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent.

[0079] In another aspect, the present disclosure provides an isolated nucleic acid encoding any one of the antigen-binding molecules described above or any one of the isolated antibodies described above.

[0080] In another aspect, the disclosure further provides a host cell comprising the above-described nucleic acid.

[0081] In another aspect, the present disclosure further provides a method for treating a disease, comprising administering to a subject a therapeutically effective amount of any one of the antigen-binding molecules described above or any one of the isolated antibodies or compositions thereof described above.

[0082] In another aspect, the present disclosure further provides the use of any one of the above described antigen-binding molecules or any one of the above described isolated antibodies or compositions thereof in the preparation of a medicament for treating a disease.

[0083] In another aspect, the present disclosure further provides an antigen-binding molecule or an isolated antibody or composition thereof according to any one of the above for use as a medicament. In some embodiments, the medicament is used for the treatment or prevention of a disease.

[0084] In some embodiments, the disease according to any one of the above is a proliferative disease (including tumors, cancer) or an autoimmune disease.

[0085] In some embodiments, the disease described in any one of the above is a hematological malignancy or a solid tumor. In some embodiments, the hematological malignancy is multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy (MGUS) (e.g., monoclonal gammopathy of undetermined diagnostic significance), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, plasmacytoplasmic leukemia, pulmonary leukemia, pulmonary fibrosis ... In some embodiments, the solid tumor is selected from the group consisting of ovarian cancer, lung cancer (non-small cell lung cancer and small cell lung cancer), gastric cancer, prostate cancer, renal cancer, liver cancer, colorectal cancer (e.g., colon cancer and rectal cancer), esophageal cancer (esophageal cancer), bladder cancer, cervical cancer, and melanoma.

[0086] In some embodiments, the disease is multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy (MGUS) (e.g., monoclonal gammopathy of undetermined diagnostic significance), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, plasma cell leukemia, light chain amyloidosis (AL), precursor B-cell lymphoblastic leukemia, leukemia, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic lymphocytic leukemia (CLL), B-cell malignancies, chronic myeloid leukemia (CML), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm, Hodgkin's lymphoma, non-Hodgkin's lymphoma, marginal zone B-cell lymphoma (MZBL), mucosa-associated lymphoid tissue lymphoma (MALT), plasma cell leukemia, anaplastic large cell lymphoma (ALCL), ovarian cancer, lung cancer, gastric cancer, prostate cancer, renal cancer, liver cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, or melanoma.

[0087] In some embodiments, the disease is multiple myeloma.

[0088] In some embodiments, the disease is relapsed / refractory multiple myeloma.

[0089] In some embodiments, the disease is a GPRC5D-associated disease, and in some embodiments, the disease is a GPRC5D-expressing disease.

[0090] The antigen-binding molecules provided in the present disclosure are characterized by high affinity, in vitro killing activity, therapeutic activity and safety. [Brief description of the drawings]

[0091] [Figure 1A] The structural schematic diagram of formula 2 is shown. [Figure 1B] The structural schematic diagram of formula 2-0G4S is shown. [Figure 1C] The structural schematic diagram of formula 4 is shown. [Figure 1D] The structural schematic diagram of formula 4-0G4S is shown. [Figure 1E] The structural schematic of formula 11 is shown. [Diagram 2] 1 shows the efficacy of antibody F2-1 in an NCIH929 subcutaneously transplanted tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] term The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0093] Unless otherwise indicated by the context, throughout the specification and claims, the words "comprise", "have", "contain" and the like should be understood to have an inclusive meaning, i.e., "including but not limited to", rather than an exclusive or exhaustive meaning. Unless otherwise stated, "comprise" includes "consists of". For example, GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 expressly covers GPRC5D-HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 1.

[0094] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0095] The term "and / or", e.g., "X and / or Y", should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both meanings or either meaning.

[0096] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in the same manner as naturally occurring amino acids. Naturally occurring amino acids are those amino acids that are coded for by codons, and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α carbon attached to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that act in the same manner as a naturally occurring amino acid.

[0097] The term "amino acid mutation" includes amino acid substitution, deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, provided that the final construct has the desired properties, such as reduced binding to Fc receptors. Deletions and insertions in the amino acid sequence include deletions and insertions at the amino and / or carboxy termini of the polypeptide chain. A specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structure and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods of modifying amino acid side groups other than genetic engineering, such as chemical modification, are also available. As used herein, various names may be used to refer to the same amino acid mutation. As used herein, the amino acid residue at a particular site may be designated by the position + amino acid residue, e.g., 366W indicates that the amino acid residue at site 366 is W. T366W indicates that the amino acid residue at site 366 has been mutated from the original T to W.

[0098] The term "antigen-binding molecule" is used in the broadest sense as long as it exhibits the desired antigen-binding activity, and covers various molecules that specifically bind to antigens, including, but not limited to, antibodies, other polypeptides having antigen-binding activity, and antibody fusion proteins formed by fusing both. The antigen-binding molecule of the present specification includes a variable region (VH) and a variable region (VL) that together constitute an antigen-binding domain. Exemplarily, the antigen-binding molecule of the present specification is a bispecific antigen-binding molecule (e.g., a bispecific antibody).

[0099] The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), provided that they exhibit the desired antigen-binding activity. For example, a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has one variable region (VH), also called the variable heavy chain domain, heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has one variable region (VL), also called the variable light chain domain or light chain variable domain, followed by one constant light chain domain (light chain constant region, CL).

[0100] A "bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, e.g., a single-chain antibody) capable of specifically binding to two different antigens or at least two different antigen epitopes of the same antigen. In the prior art, bispecific antibodies of various structures are disclosed, which are divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies according to the integrity of the IgG molecule, bispecific antibodies having two, three, four or more valences according to the number of antigen-binding regions, and bispecific antibodies having a symmetric structure and bispecific antibodies having an asymmetric structure according to whether the structure is symmetric. Here, the bispecific antibody based on antibody fragments is, for example, a Fab fragment lacking an Fc fragment, and two or more Fab fragments are bound to one molecule to form a bispecific antibody, which has relatively low immunogenicity, a small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type are, for example, F(ab)2, scFv-Fab, and (scFv)2-Fab. Antibodies such as IgG-like bispecific antibodies (e.g., having an Fc fragment) have a relatively large molecular weight, and the Fc fragment contributes to the purification of the antibody and improves its solubility and stability. The Fc portion can also bind to the receptor FcRn to increase the half-life of the antibody serum. Typical structural models of bispecific antibodies include, for example, KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (Aran See F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages585~608 (2019), Chen S1 et al., J Immunol Res.2019 Feb 11, 2019:4516041).

[0101] The term "variable region" or "variable domain" refers to a domain that binds to an antigen in an antigen-binding molecule. In this specification, the heavy chain variable region of an antigen-binding module that specifically binds to GPRC5D is represented as GPRC5D-VH and the light chain variable region is represented as GPRC5D-VL, and the heavy chain variable region of an antigen-binding module that specifically binds to CD3 is represented as CD3-VH and the light chain variable region is represented as CD3-VL. VH and VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDRs). Among them, the term "complementarity determining region" or "CDR" refers to the region that mainly promotes binding to an antigen in the variable domain, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. In this specification, the three CDR regions in GPRC5D-VH are represented as GPRC5D-HCDR1, GPRC5D-HCDR2 and GPRC5D-HCDR3, the three CDR regions in GPRC5D-VL are represented as GPRC5D-LCDR1, GPRC5D-LCDR2 and GPRC5D-LCDR3, the three CDR regions in CD3-VH are represented as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, and the three CDR regions in CD3-VL are represented as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3. Each VH and VL is represented as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus. A single VH or VL can be sufficient to confer antigen binding specificity.

[0102] The amino acid sequence boundaries of the CDRs can be determined by various known methods, such as, for example, the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), Front Immunol. 2018 Oct 16; 9: 2278), and the correspondence between various numbering systems is well known to those of skill in the art. The numbering conventions of the present disclosure are as shown in Table 1 below.

[0103] [Table 1]

[0104] Unless otherwise stated, all variable region and CDR sequences in the examples of this disclosure follow the "Kabat" numbering convention.

[0105] The term "antibody fragment" refers to a molecule distinct from a complete antibody, which includes a portion of the complete antibody that retains the antigen-binding ability of the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and multispecific antibodies composed of antibody fragments.

[0106] As described herein, the terms "Fab" or "Fab fragment" may be used interchangeably and refer to a monovalent antibody fragment consisting of VH, VL, CH1 and CL domains, and as used herein, a Fab monovalent antibody fragment of an antibody such as an anti-GPRC5D antibody can be linked to an Fc1 subunit at the C-terminus of its CH1 directly or via a linker, and the formed Fab-Fc1 can further form a bispecific antibody with Fab-Fc2 of another antibody such as an anti-CD3 antibody via a disulfide bond at the N-terminus of the Fc1 subunit.

[0107] As used herein, the term "scFv" refers to a single-chain variable fragment in which an antibody heavy chain variable region (VH) and light chain variable region (VL) are linked by a flexible short peptide (linker) consisting of 10 to 25 amino acids, and is the smallest form of a recombinant antibody.

[0108] The term "Fc region" or "fragment crystallizable region" is intended to define the C-terminal region of an antibody heavy chain, and includes native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be identical or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226, or from Pro230, to its carboxy terminus. Suitable native sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU index.

[0109] The term "Titin chain" refers to a peptide fragment having a length of 78 to 118 amino acids in the Titin protein and containing the Titin Ig-Like 152 domain, or a functional variant thereof, and the Titin chain can bind to Obscurin Ig-Like 1 or Obscurin-Like Ig-Like 1 domain to form a dimerization complex. The term "Obscurin chain" refers to a peptide fragment having a length of 87 to 117 amino acids in the Obscurin protein and containing the Obscurin Ig-Like 1 domain, or a functional variant thereof, and a peptide fragment having a length of 78 to 118 amino acids in the Obscurin-Like 1 protein and containing the Obscurin-Like Ig-Like 1 domain, or a functional variant thereof, and the Obscurin chain can bind to the Titin Ig-Like 152 domain to form a dimerization complex. The Titin chain and Obscurin chain of the present disclosure can be used to replace CH1 and CL in a Fab to form a substituted Fab (Fab-S), which does not affect the binding of the antigen-binding molecule to an antigen.

[0110] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0111] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while at the same time having relatively low immunogenicity in humans, which may be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (i.e., the constant and variable region framework region portions).

[0112] The term "affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to internal binding affinity and reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can usually be expressed as an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art (as described herein). The term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while "kdis" or "kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed in molar concentration (M). The KD value of an antibody can be measured by methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).

[0113] The term "monoclonal antibody" refers to a population of essentially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules in the population are the same, except for minor natural mutations that may exist. In contrast, a polyclonal antibody preparation usually contains a number of different antibodies having different amino acid sequences in their variable domains that are generally specific for different epitopes. "Monoclonal" characterizes the antibody as being obtained from an essentially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0114] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent of an antigen-binding protein (e.g., an antibody). An antigen may have one or more epitopes that are capable of interacting with different antigen-binding proteins (e.g., antibodies).

[0115] The term "epitope" refers to an area or region on an antigen capable of specific binding to an antibody or antigen-binding fragment thereof. An epitope may be formed from contiguous amino acids (linear epitope) or may comprise non-contiguous amino acids (conformational epitope), for example, where non-contiguous amino acids are brought into spatial proximity by folding of the antigen (i.e., tertiary folding of a proteinaceous antigen). Conformational and linear epitopes differ in that antibody binding to conformational epitopes is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed by methods routine in the art, including, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide truncation analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0116] The terms "capable of specifically binding," "specifically binds," or "binding" refer to the ability of an antibody to bind to an antigen or epitope with greater affinity than to other antigens or epitopes. Generally, antibodies bind to antigens or epitopes with greater affinity than other antigens or epitopes with greater affinity than other antigens or epitopes. -7 M or less (for example, about 1×10 -8An antibody binds to an antigen or epitope with an equilibrium dissociation constant (KD) of 0.1 M or less. In some embodiments, the KD of an antibody to an antigen is 10% or less (e.g., 1%) of the KD of the antibody to a non-specific antigen (e.g., BSA, casein). KD can be measured by known methods, for example, FACS or surface plasmon resonance assay. However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, cross-reactivity to corresponding antigens from other species (homo) (e.g., humans or monkeys such as Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp)) or Callithrix jacchus (common marmoset, marmoset).

[0117] The term "does not bind" means that the antibody is unable to bind to an antigen or an epitope within the antigen in the manner of specific binding described above. For example, the antibody may be capable of binding to an antigen having a specific binding activity of about 1×10 -6 It binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of greater than or equal to M.

[0118] The term "antigen binding module" refers to a polypeptide molecule that specifically binds to a target antigen. A specific antigen binding module includes, for example, an antigen binding domain of an antibody, including a heavy chain variable region and a light chain variable region. The term "antigen binding module that specifically binds to GPRC5D" refers to a module that can bind to GPRC5D with sufficient affinity so that a molecule that includes the module can be used as a diagnostic and / or therapeutic agent targeting GPRC5D. For example, an antigen binding module that specifically binds to GPRC5D has an equilibrium dissociation constant (KD) that is < about 10 nM, as measured by a surface plasmon resonance assay. An antigen binding module includes an antibody fragment, such as a Fab, a substituted Fab, or a scFv, as defined herein.

[0119] The term "linker" refers to a linking unit that links two polypeptide fragments. In this specification, linkers appearing in the same structural formula may be the same or different. The linker may be a peptide linker and comprises one or more amino acids, typically comprising about 1-30, 2-24, or 3-15 amino acids. The linkers used in this specification may be the same or different. When "-" appears in a structural formula, it indicates that the units on both sides are directly linked by a covalent bond.

[0120] "Tm" is the melting denaturation temperature (intrinsic fluorescence). When a protein is denatured (by heating or by the action of a denaturing agent), the tertiary structure opens and the microenvironment of aromatic amino acids is changed, which results in a change in the emission fluorescence spectrum. In this disclosure, Tm1 refers to the temperature at which the fluorescence is changed to half of its maximum value.

[0121] "Tonset" is the onset temperature of denaturation. It means the temperature at which the protein starts to denature, i.e., the temperature at which the fluorescence value starts to change.

[0122] "Tagg" is the aggregation onset temperature. The aggregates were detected by static light scattering at two wavelengths, 266 nm and 473 nm, to monitor the temperature at the onset of sample aggregation. Tagg 266 refers to the onset of aggregation temperature monitored at 266 nm.

[0123] The term "nucleic acid" may be used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term covers nucleic acids containing known nucleoside analogs or modified backbone residues or linkers, synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties as the reference nucleic acid and are metabolized in the same manner as the reference nucleoside. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell, as described below, which generally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. The isolated nucleic acid encoding the antigen-binding molecule refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including one or more such nucleic acid molecules contained in a single vector or separate vectors, and one or more such nucleic acid molecules present at one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence implicitly covers its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence specified. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating a sequence in which the third position of one or more selected (or all) codons is replaced with a degenerate base and / or a deoxyinosine residue.

[0124] The terms "polypeptide" and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, and apply to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0125] The term sequence "identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned. During alignment, gaps can be introduced as necessary to obtain the maximum percentage of sequence identity, and any conservative substitutions are not considered as part of the sequence identity. To measure the percentage of sequence identity, alignment can be achieved by techniques known in the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0126] The terms "fused" or "linked" refer to covalent linkage of elements (e.g., an antigen binding module and an Fc domain) either directly or via a linker.

[0127] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA circle that can be ligated to an additional DNA segment. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which another DNA segment can be ligated to the viral genome. Some vectors can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector that is useful for transformation of a host cell and contains (in conjunction with the host cell) nucleic acid sequences that direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, as well as RNA splicing of the coding region operably linked to introns, if any.

[0128] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its derived progeny, without regard to the number of passages. The progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. As used herein, the term includes mutant progeny that have the same function or biological activity as the cell screened or selected from the original transformed cell. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells, including, but not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, and the like. stiptis, Pichia methanolica, Pichia spp., Saccharomyces cerevisiae, Saccharomyces spp., Hansenula polymorpha, Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium spp. sp.), Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, Neurospora crassa, Aspergillus spp., and Yarrowia lipolytica.Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa. Host cells in this patent do not include those not permitted by patent law.

[0129] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is further understood that any progeny may not have exactly the same DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as the initially transformed cell for which they were screened are included.

[0130] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but does not necessarily occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0131] The term "pharmaceutical composition" refers to a mixture containing one or more antigen-binding molecules or antibodies described herein and other chemical components, such as physiologically / pharmaceutical acceptable vectors and excipients.

[0132] The term "pharmaceutically acceptable vector" refers to a component in a drug formulation that is different from the active ingredient and is non-toxic to a subject. Pharmaceutically acceptable vectors include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0133] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the above terms "patient" or "subject" may be used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to cynomolgus monkeys (Macaca fascicularis). In some embodiments, the individual or subject is a human.

[0134] "Administration" or "giving" as applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid means contact of an exogenous agent, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid.

[0135] The term "sample" refers to collections similar to fluids, cells, or tissues isolated from a subject, and fluids, cells, or tissues present within the body of a subject. Exemplary samples are biological fluids, such as blood, serum and serous fluid, plasma, lymphatic fluid, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal and other body cavities fluids, fluids collected from bronchial washings, synovial fluid, liquid solutions that have come into contact with a subject or biological source, such as cell and organ media (including cell or organ conditioned media), lavage fluids, tissue biopsies, fine needle aspirates, surgically excised tissues, organ cultures, or cell cultures.

[0136] "Treatment" and "treatment" (and grammatical variations thereof) refer to a clinical intervention intended to be given to the individual being treated, and can be performed prophylactically or in the clinical pathological process. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction / reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or amelioration of the disease state, and regression or improvement of prognosis. In some embodiments, the molecules of the present disclosure are used to delay the formation of disease or to slow the progression of disease.

[0137] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or underlying causes, prevent the appearance of symptoms and / or their underlying causes, and / or improve or ameliorate damage caused by or associated with a disease state (e.g., pulmonary disease). In some embodiments, the effective amount is a therapeutically or prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a condition or symptom associated with the disease state, or to prevent, inhibit, delay or reverse the progression of the disease state or any other undesirable symptoms associated in any manner with the disease state or the disease in any manner. A "prophylactically effective amount" is an amount that, when administered to a subject, provides a desired prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or prophylactic effect does not necessarily occur when one dose is administered, but may occur after a series of doses are administered. Thus, a therapeutically or prophylactically effective amount can be administered in a single or multiple dose regime. A "therapeutically effective amount" and a "prophylactically effective amount" can vary depending on a variety of factors, such as, for example, the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indications of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient. Antigen-binding molecules of the present disclosure

[0138] The present disclosure provides antigen-binding molecules that have many advantageous properties, such as high affinity for GPRC5D and GPRC5D-expressing cells, in vitro killing activity, therapeutic activity and safety. Exemplary antigen-binding molecules

[0139] The antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (e.g., bispecific antibodies) and anti-GPRC5D antibodies that specifically bind to GPRC5D and CD3. In particular, the antigen-binding molecules of the present disclosure have any one of the following properties: a. High affinity for GPRC5D, in some embodiments, with an EC of less than 50 nM (e.g., less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 2.5 nM, less than 1 nM, less than 0.5 nM). 50 and / or has an EC 50 It has good cross-linking activity against CynoGPRC5D at the above EC 50 is detected by flow cytometry; b. It does not bind to human CCR8 and human GIPR and has good specificity; c. In vitro specific killing activity against GPRC5D expressing cells; d. Inducing the release of low levels of cytokines (IL6 and IFNγ); and / or e. More potent in vivo therapeutic activity.

[0140] The present disclosure provides an antigen-binding molecule, which comprises at least one antigen-binding module that specifically binds to GPRC5D and at least one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to GPRC5D comprises GPRC5D-VH and GPRC5D-VL, and the antigen-binding module that specifically binds to CD3 comprises CD3-VH and CD3-VL.

[0141] The present disclosure further provides an isolated antibody capable of specifically binding to GPRC5D, the antibody comprising GPRC5D-VH and GPRC5D-VL. Specifically, the examples herein disclose antibody series 1, 24, 30 and 45. Hereinafter, the antibodies of the present disclosure will be described taking antibody 1 and 24 as examples.

[0142] An antigen-binding molecule or anti-GPRC5D antibody that specifically binds to GPRC5D and CD3, wherein the GPRC5D-VH has GPRC5D-HCDR1 having the amino acid sequence of SEQ ID NO: 9, GPRC5D-HCDR2 having the amino acid sequence of SEQ ID NO: 134, and GPRC5D-HCDR3 having the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has GPRC5D-LCDR1 having the amino acid sequence of SEQ ID NO: 130, GPRC5D-LCDR2 having the amino acid sequence of SEQ ID NO: 13, and GPRC5D-LCDR3 having the amino acid sequence of SEQ ID NO: 14.

[0143] An antigen-binding molecule or anti-GPRC5D antibody that specifically binds to GPRC5D and CD3, wherein the GPRC5D-VH has GPRC5D-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 9, GPRC5D-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 134, and GPRC5D-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 11, and also has GPRC5D-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 130, GPRC5D-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 13, and GPRC5D-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 14.

[0144] An antigen-binding molecule or anti-GPRC5D antibody that specifically binds to GPRC5D and CD3, wherein the GPRC5D-VH has GPRC5D-HCDR1 having the amino acid sequence of SEQ ID NO: 15, GPRC5D-HCDR2 having the amino acid sequence of SEQ ID NO: 16, and GPRC5D-HCDR3 having the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has GPRC5D-LCDR1 having the amino acid sequence of SEQ ID NO: 137, GPRC5D-LCDR2 having the amino acid sequence of SEQ ID NO: 19, and GPRC5D-LCDR3 having the amino acid sequence of SEQ ID NO: 20.

[0145] An antigen-binding molecule or anti-GPRC5D antibody that specifically binds to GPRC5D and CD3, wherein the GPRC5D-VH has GPRC5D-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 15, GPRC5D-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 16, GPRC5D-HCDR3 whose amino acid sequence is shown in SEQ ID NO: 17, GPRC5D-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 137, GPRC5D-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 19, and GPRC5D-LCDR3 whose amino acid sequence is shown in SEQ ID NO: 20.

[0146] In some embodiments, in the antigen-binding molecule or antibody, the GPRC5D-VH and / or the GPRC5D-VL are murine or humanized.

[0147] In some embodiments, the GPRC5D-VH and / or the GPRC5D-VL are humanized.

[0148] In some embodiments, the GPRC5D-VH has FR1, FR2, FR3 from IGHV3-21*01 and FR4 from IGHJ6*01, which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 49A and 93T, and / or the GPRC5D-VL has FR1, FR2, FR3 from IGKV2-29*02 and FR4 from IGKJ2*01, which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 2V and 45K. In some embodiments, the variable regions and CDRs are defined according to the Kabat numbering convention.

[0149] In some embodiments, the antigen-binding molecule or antibody described in any one of the above, wherein the amino acid sequence of the GPRC5D-VH has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 35, 1, 33 or 34, and the amino acid sequence of the GPRC5D-VL has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41.

[0150] In some embodiments, the GPRC5D-VH and / or the GPRC5D-VL are humanized.

[0151] In some embodiments, the GPRC5D-VH has FR1, FR2, FR3 from IGHV1-46*01 and FR4 from IGHJ6*01, which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 24V, 37M, 69W, 71A, 73K and 93S, and / or the GPRC5D-VL has FR1, FR2, FR3 from IGKV2-30*02 and FR4 from IGKJ4*01, which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 4L, 28T, 28S, 29L, 29V, 36L and 37L. In some embodiments, the variable regions and CDRs are defined according to the Kabat numbering convention.

[0152] In some embodiments, the antigen-binding molecule or antibody is any one of the above, wherein the amino acid sequence of the GPRC5D-VH has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 44, 3, 42 or 43, and the amino acid sequence of the GPRC5D-VL has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50.

[0153] In some embodiments, the amino acid sequence of the GPRC5D-VH is represented by SEQ ID NO: 35, 1, 33 or 34, and the amino acid sequence of the GPRC5D-VL is represented by SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41.

[0154] In some embodiments, the amino acid sequence of the GPRC5D-VH is represented by SEQ ID NO: 35, 33 or 34, and the amino acid sequence of the GPRC5D-VL is represented by SEQ ID NO: 39, 36, 37, 38, 40 or 41.

[0155] In some embodiments, the amino acid sequence of the GPRC5D-VH is represented by SEQ ID NO: 44, 3, 42 or 43, and the amino acid sequence of the GPRC5D-VL is represented by SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50.

[0156] In some embodiments, the amino acid sequence of the GPRC5D-VH is represented by SEQ ID NO: 44, 42 or 43, and the amino acid sequence of the GPRC5D-VL is represented by SEQ ID NO: 49, 45, 46, 47, 48 or 50.

[0157] In some embodiments, the antigen-binding molecule or antibody according to any one of the above, The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 1, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 2; Antigen-binding molecules.

[0158] In some embodiments, the antigen-binding molecule or antibody is any one of the above, wherein the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39.

[0159] In some embodiments, the antigen-binding molecule or antibody is any one of the above, wherein the amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 35, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 39.

[0160] In some embodiments, the antigen-binding molecule or antibody according to any one of the above, The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 3, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 4; An antigen-binding molecule or antibody.

[0161] In some embodiments, the antigen-binding molecule or antibody is any one of the above, wherein the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49.

[0162] In some embodiments, the antigen-binding molecule or antibody is any one of the above, wherein the amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 44 and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 49.

[0163] In some embodiments, the antigen-binding molecule described above, wherein the CD3-VH has a CD3-HCDR1 whose amino acid sequence is set forth in SEQ ID NO: 76, a CD3-HCDR2 whose amino acid sequence is set forth in SEQ ID NO: 77, and a CD3-HCDR3 whose amino acid sequence is set forth in SEQ ID NO: 78, and the CD3-VL has a CD3-LCDR1 whose amino acid sequence is set forth in SEQ ID NO: 79, a CD3-LCDR2 whose amino acid sequence is set forth in SEQ ID NO: 80, and a CD3-LCDR3 whose amino acid sequence is set forth in SEQ ID NO: 81.

[0164] In some embodiments, the antigen binding molecule of any one of the above, wherein the CD3-VH and / or the CD3-VL is murine or humanized.

[0165] In some embodiments, the CD3-VH and / or the CD3-VL are humanized.

[0166] In some embodiments, the amino acid sequence of the CD3-VH is set forth in SEQ ID NO:82, and the amino acid sequence of the CD3-VL is set forth in SEQ ID NO:83.

[0167] In some embodiments, the variable regions and CDRs are defined according to the Kabat numbering convention.

[0168] According to the technical solutions of antibody series 30 and 45 disclosed in the examples of this specification, these antibodies have similar technical solution scope as antibodies 1 and 24 mentioned above. Structure of antigen-binding molecules

[0169] The bispecific antigen-binding molecule of the present disclosure is not limited to a specific molecular structure as long as it has the desired antigen-binding function. For example, the bispecific antigen-binding molecule of the present disclosure may be bivalent (1+1), trivalent (2+1), or tetravalent (2+2). The antigen-binding module in the antigen-binding molecule may be any antibody fragment having antigen-binding activity fused via a peptide linker. The peptide linker of the present disclosure (e.g., linkers 1-6) may be any suitable peptide chain as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide of 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have a structure of L1-(GGGGS)n-L2, in which L1 is a bond, A, GS, GGS, GGGS (SEQ ID NO: 213), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG (SEQ ID NO: 95), or GGGG (SEQ ID NO: 214), and the peptide linker is not a bond. In some embodiments, the length of the peptide linker is 3 to 15 amino acid residues. In some embodiments, the peptide linkers each independently have an amino acid sequence of (GGGGS)n, in which n is 1 to 5, preferably n is 1, 2, or 3. In some embodiments, the peptide linker is GGGGS (SEQ ID NO: 90), GGGGSGGGSGGGGS (SEQ ID NO: 94), or GGG (SEQ ID NO: 95). In some embodiments, the amino acid sequences of linker 1, linker 2, linker 3 and linker 4 are set forth in SEQ ID NO: 90, the amino acid sequence of linker 5 is set forth in SEQ ID NO: 94, and the amino acid sequence of linker 6 is set forth in SEQ ID NO: 95.

[0170] Exemplarily, the antigen-binding molecule of the present disclosure comprises one first chain having a structure represented by formula (i), one second chain having a structure represented by formula (j), one third chain having a structure represented by formula (k-1), and one fourth chain having a structure represented by formula (l-1); (a) [GPRC5D-VH]-[CH1]-[Fc1], (b) [GPRC5D-VL]-CL, (g-1)[CD3-VH]-[GGGGS]-[Obscurin chain]-[Fc2], (h-1)[CD3-VL]-[GGGGS]-[Titin chain], The structures represented by formulae (a), (b), (g-1) and (h-1) are arranged from the N-terminus to the C-terminus.

[0171] For example, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 118, one second chain comprising the amino acid sequence of SEQ ID NO: 102, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 120, one second chain comprising the amino acid sequence of SEQ ID NO: 106, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 110, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 124, one second chain comprising the amino acid sequence of SEQ ID NO: 117, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85. Mutants of antigen-binding molecules

[0172] In some embodiments, amino acid sequence variants of the antigen-binding molecules provided herein are covered. For example, it is desired to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antigen-binding molecule. The final construct can be obtained by any combination of deletion, insertion and substitution, provided that the final construct has the desired properties, such as antigen-binding properties. Mutant substitutions, insertions and deletions

[0173] In one embodiment, variants of antigen-binding molecules are provided that have one or more amino acid substitutions. Substitutions can be made in the CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". More substantial changes are provided in Table 2 under the heading of "exemplary substitutions" and are as further described below with reference to amino acid side chain classifications. Amino acid substitutions can be introduced into an antibody of interest and the product screened for desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0174] [Table 2]

[0175] Amino acids can be grouped according to common side chain properties as follows: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0176] Non-conservative substitutions refer to the replacement of a member of one class with a member of another class.

[0177] One type of substitution variant involves the substitution of one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study has an altered (e.g., improved) biological property (e.g., improved affinity, reduced immunogenicity) relative to the parent antibody and / or essentially retains some biological property of the parent antibody. One exemplary substitution variant is an affinity matured antibody, which can be readily produced, for example, by phage display-based affinity maturation techniques (e.g., techniques as described herein). Briefly, one or more CDR residues are mutated, the mutated antibodies are displayed on phage, and screened for a particular biological activity (e.g., binding affinity). For example, changes (e.g., substitutions) can be made in the CDRs to improve antibody affinity. Such changes can be made to CDR "hot spots", i.e., residues encoded by codons frequently mutated during the somatic maturation process and / or residues that contact the antigen, and the resulting variant VH or VL can be tested for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves CDR-targeting, where a few CDR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify CDR residues involved in antigen binding.

[0178] In certain embodiments, substitutions, insertions or deletions may be made in one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) can be made to the CDRs that do not substantially reduce binding affinity. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two or three amino acid substitutions.

[0179] A method that can be used to identify residues or regions that can be target sites for mutagenesis in an antibody is called "alanine scanning mutagenesis". In this method, a residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to see if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Also, contact points between the antibody and the antigen can be identified by examining crystal structures of antigen-antibody complexes. These contact and adjacent residues can be targeted or removed as substitution candidates. Mutants can be screened to see if they contain the desired properties.

[0180] Amino acid sequence insertions include the fusion of polypeptides of one residue or 100 or more residues in length at the amino and / or carboxyl terminus, and the insertion of one or more amino acid residues into the sequence. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions to the N- or C-terminus of the antibody with an enzyme (or a polypeptide which extends the serum half-life of the antibody). Fab modification

[0181] In one embodiment, in the antigen-binding molecule of the present disclosure, one of the antigen-binding module that specifically binds to GPRC5D and the antigen-binding module that specifically binds to CD3 is a substituted Fab containing a heavy chain variable region, a light chain variable region, a Titin chain, and an Obscurin chain. In the substituted Fab, the original CH1 and CL of the Fab are replaced with the Titin chain and the Obscurin chain (for example, the original CH1 and CL of the Fab are replaced with the Obscurin chain and the Titin chain, respectively, or the original CH1 and CL of the Fab are replaced with the Titin chain and the Obscurin chain, respectively). Exemplarily, the sequences of the Titin chain and the Obscurin chain are as shown in Table 3-1 and Table 3-2.

[0182] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 4-3] Fc Region Modification

[0183] In one aspect, the Fc region of the antigen binding molecule of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an Fcγ receptor, and reduce or eliminate effector function. A native IgG Fc region is specifically an IgG1 Fc region or an IgG4 Fc region, which may allow the antigen binding molecule of the present disclosure to target Fc receptor-expressing cells rather than antigen-expressing cells. The modified Fc region of the present disclosure exhibits reduced binding affinity to an Fc receptor and / or reduced effector function. In some embodiments, the modified Fc region has a reduced binding affinity to an Fc receptor of 50%, 80%, 90%, or 95% or more compared to the native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the altered Fc region also has reduced binding affinity to complement, such as C1q, compared to the native Fc region. In some embodiments, the altered Fc region does not have reduced binding affinity to the neonatal Fc receptor (FcRn) compared to the native Fc region. In some examples, the altered Fc region has reduced effector function, which may include, but is not limited to, one or more of reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen presenting cell uptake, reduced binding to NK cells, reduced binding to macrophage cells, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced cell apoptosis, reduced dendritic cell maturation, or reduced triggering by T cells. For the IgG1 Fc region, substitution of amino acid residues at positions such as 238, 265, 269, 270, 297, 327 and 329 can reduce effector functions.In some embodiments, the Fc region is a human IgG1 Fc region and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For IgG4 Fc regions, substitution of amino acid residues at positions such as 228 can reduce effector function.

[0184] The antigen-binding molecule may further include disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit. To increase the serum half-life of the antigen-binding molecule, the following mutations can be introduced: 252Y, 254T, and 256E.

[0185] When an antigen-binding molecule contains different binding modules fused to two subunits in the Fc region, this may lead to undesired homodimerization. To improve yield and purity, it is advantageous to introduce heterodimerization-promoting modifications to the Fc region of the antigen-binding molecule of the present disclosure. In some embodiments, the Fc region of the present disclosure includes a knob-into-hole (KIH) technique modification, which involves introducing a knob structure at the interface of the first subunit and a hole structure at the interface of the second subunit. This allows the knob structure to be positioned at the hole structure, promoting the formation of heterodimers and inhibiting the production of homodimers. The knob structure is constructed by replacing a small amino acid side chain from the interface of the first subunit with a relatively large side chain (e.g., tyrosine or tryptophan). Meanwhile, the hole structure is constructed at the interface of the second subunit by replacing a large amino acid side chain with a relatively small amino acid side chain (e.g., alanine or threonine). The knob and hole structures are prepared by altering the nucleic acid encoding the polypeptide, with selectable amino acid substitutions as shown in the table below.

[0186] [Table 5]

[0187] Except for the knobs-in-holes technique, other techniques for modifying the CH3 domain of the heavy chain so as to achieve heterodimerization are also known in the art, e.g. WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954 and WO013 / 096291.

[0188] The C-terminus of the Fc region may be a complete C-terminus terminating in amino acid residue PGK, or may be a truncated C-terminus, e.g., one or two C-terminal amino acid residues removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus terminating in PG. Thus, in some embodiments, a composition of complete antibodies may comprise antibodies in which any K447 residue and / or G446+K447 residues have been removed. In some embodiments, a composition of complete antibodies may comprise antibodies in which the K447 residue and / or G446+K447 residues have not been removed. In some embodiments, a composition of complete antibodies comprises antibodies of an antibody mixture with and without the K447 residue and / or the G446+K447 residues. Recombinant methods

[0189] Antigen-binding molecules can be produced by recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecule are provided.

[0190] In the case of a natural antibody, a natural antibody fragment, or a bispecific antibody with a homodimeric heavy chain, two nucleic acids are required: one for the light chain or a fragment thereof, and one for the heavy chain or a fragment thereof. Such nucleic acids encode an amino acid sequence comprising the antibody VL and / or an amino acid sequence comprising the antibody VH (e.g., the light and / or heavy chains of the antibody). These nucleic acids can be present in the same expression vector or in different expression vectors.

[0191] In the case of a bispecific antibody having a heterodimeric heavy chain, for example, four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising a first heterologous monomeric Fc region polypeptide, one for the second light chain and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. The four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors; typically, these nucleic acids are located in two or three expression vectors, i.e., one vector may contain one or more of these nucleic acids.

[0192] In one embodiment, the disclosure provides an isolated nucleic acid encoding the antibody. Each such nucleic acid can independently encode any one of the polypeptide chains. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In another aspect, the disclosure provides a host cell comprising such a nucleic acid. In one embodiment, a method of preparing an antigen-binding molecule is provided, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell medium).

[0193] To produce an antigen-binding molecule recombinantly, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be easily isolated and sequenced by routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an antibody), or produced by recombinant methods, or obtained by chemical synthesis.

[0194] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. After expression, antibodies can be isolated from bacterial cell paste in a soluble fraction and can be further purified.

[0195] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns. Suitable host cells for the expression (glycosylation) of antibodies may be derived from multicellular organisms (invertebrates and vertebrates), examples of invertebrate cells include plants and insect cells. Many baculovirus strains have already been identified that may be used in conjunction with insect cells, in particular for transfection of Spodoptera frugiperda cells, and plant cell cultures such as US5959177, US6040498, US6420548, US7125978 and US6417429 may also be used as hosts, as well as vertebrate cells, e.g. mammalian cell lines suitable for growth in suspension. Other examples of suitable mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, MRC 5 cells and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of several mammalian host cell lines suitable for the production of antibodies, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268. immune complex

[0196] The present disclosure further provides an immunoconjugate comprising an antigen-binding molecule conjugated to one or more cytotoxic agents, the one or more cytotoxic agents being, for example, a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant or animal origin, an enzymatically active toxin or fragment thereof), or a radioisotope. Diagnostic and Therapeutic Compositions

[0197] In some embodiments, the antigen-binding molecules provided herein can be used to detect the presence of GPRC5D and / or CD3 in a biological sample. As used herein, the term "detection" covers quantitative or qualitative detection. In some embodiments, the biological sample includes cells or tissues such as tumor tissues.

[0198] In one embodiment, the antigen-binding molecule is used in a diagnostic or detection method. In another aspect, a method for detecting the presence of GPRC5D and / or CD3 in a biological sample is provided. In one embodiment, the method comprises contacting a biological sample with an antigen-binding molecule under appropriate conditions and detecting whether a complex is formed between the detection agent and the antigen. Such a method may be an in vitro method or an in vivo method. In one embodiment, the antigen-binding molecule is used to select subjects suitable for treatment, for example, GPRC5D and / or CD3 are biomarkers for selecting patients.

[0199] Exemplary disease states that can be diagnosed using the antigen binding molecules of the present disclosure are, for example, tumors and cancers.

[0200] In some embodiments, a labeled antigen-binding molecule is provided. Labels include, but are not limited to, directly detected labels or modules (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent and radioactive labels) and indirectly detected modules (e.g., modules indirectly detected by enzymatic reactions or molecular interactions, e.g., enzymes or ligands).

[0201] In another embodiment, a pharmaceutical composition comprising the antigen-binding molecule is provided, for example, for use in any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the antigen-binding molecules provided herein and a pharma- ceutical acceptable vector. In another embodiment, the pharmaceutical composition comprises any of the antibodies provided herein and at least one other therapeutic agent.

[0202] The pharmaceutical composition of the antigen-binding molecule described in the present disclosure is prepared by mixing such an antigen-binding molecule having the required purity with one or more pharma- ceutically acceptable vectors, and the pharmaceutical composition is in the form of a lyophilized composition or an aqueous solution. Preparations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. Treatment Methods and Routes of Administration

[0203] Any of the antigen binding molecules provided herein can be applied in therapeutic methods.

[0204] In a further aspect, the disclosure provides a use of an antigen-binding molecule in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a tumor or cancer. Also, the medicament is present in an amount effective for the disease. In some embodiments, the effective amount is a daily dose or a weekly dose. In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents). The "subject" according to any of the above embodiments may be a human.

[0205] In a further aspect, there is provided a pharmaceutical composition comprising the antigen-binding molecule, for example for use in any of the pharmaceutical uses or therapeutic methods described above. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0206] The antigen-binding molecules of the present disclosure can be used alone or in combination with other reagents for treatment. For example, the antigen-binding molecules of the present disclosure can be administered together with at least one additional therapeutic agent.

[0207] The antigen-binding molecules of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, pulmonary, and intranasal administration, and, where localized treatment is required, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0208] The antigen-binding molecules of the present disclosure are formulated, dosed and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific condition being treated, the specific mammal being treated, the clinical status of the individual patient, the cause of the condition, the delivery site of the reagent, the method of administration, the administration schedule and other factors known to medical practitioners. The antigen-binding molecules may not be formulated together with one or more reagents currently used to prevent or treat the above-mentioned conditions, but may optionally be. The effective amount of such other reagents will depend on the amount of antigen-binding molecules present in the pharmaceutical composition, the type of condition or treatment, and other factors discussed above. These are generally used in the same doses and administration routes as described herein, or from about 1% to about 99% of the doses described herein, or in any dose and by any route empirically / clinically confirmed to be appropriate.

[0209] The appropriate dose of the antigen-binding molecule according to the present disclosure (used alone or in combination with one or more additional therapeutic agents) for preventing or treating a disease is determined by the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether the administration is for prevention or treatment, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is administered to the patient in one or a series of treatments, as appropriate. Depending on the type and severity of the disease, about 1 μg / kg to about 15 mg / kg of the antigen-binding molecule may be an initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dose may range from about 1 μg / kg to about 100 mg / kg or more, depending on the factors mentioned above. Thus, for a 50 kg body weight, an exemplary daily dose is 50 μg to 5 g. product

[0210] In another aspect of the disclosure, an article of manufacture is provided that includes materials applicable for treating, preventing, and / or diagnosing the above-mentioned medical conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as, for example, glass or plastic. The container may contain a composition that, alone or in combination with another composition, effectively treats, prevents, and / or diagnoses a disease, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a plug that can be pierced by a hypodermic needle). At least one active agent in the composition is an antigen-binding molecule according to the present disclosure. The label or package insert indicates that the composition is used to treat a selected medical condition. The article of manufacture may also include (a) a first container that contains a composition that includes an antigen-binding molecule according to the present disclosure, and (b) a second container that contains a composition that includes another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the disclosure may further include a package insert, the package insert indicating that the composition is applicable for treating a particular medical condition. Optionally, or in addition, the article of manufacture may further include a second (or third) container containing a pharma-ceutically acceptable buffer. From a business and user standpoint, it may further include other materials required, including other buffers, diluents, filters, needles and syringes. Examples and Tests

[0211] The present disclosure will be further described below with reference to examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples and test examples of the present disclosure generally follow standard conditions, such as those in the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual, or conditions recommended by manufacturers of raw materials or products. Reagents for which a specific source is not specified are standard commercially available reagents. Example 1 Antigen-binding molecule containing Titin chain / Obscurin chain

[0212] The Titin chain / Obscurin chain of the present disclosure may be derived from any suitable polypeptide, including those derived from WO2021139758 (incorporated herein in its entirety by reference) and CN202110527339.7, and patents which claim priority thereto (incorporated herein in their entirety by reference). A bispecific antibody was constructed, in which CL is the kappa light chain constant region in WO2021139758, the amino acid sequences of the Titin chain and Obscurin chain are shown in Tables 3-1 and 3-2, the linker sequence comprises GGGGS (SEQ ID NO: 90), ASTKG (SEQ ID NO: 209) or RTVAS (SEQ ID NO: 210), and the amino acid sequences of Fc1, Fc2, and CH1 in this example are as follows:

[0213] Example 1 - Fc1 (knob, SEQ ID NO: 92): [ka] Example 1 - Fc2 (hole, SEQ ID NO: 93): [ka] Example 1 - CH1 (SEQ ID NO: 91): [ka] 1.1 DI bispecific antibodies

[0214] With reference to Example 5 of WO2021139758, anti-hNGF and hRANKL DI bispecific antibodies: DI-2 to DI-20 were constructed, which comprise the following first heavy chain, second heavy chain, first light chain and second light chain: First heavy chain: from the N-terminus to the C-terminus, it is [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2]; First light chain: from the N-terminus to the C-terminus, it is [VL1-I]-[Linker 2]-[Titin chain]; A second heavy chain is, from the N-terminus to the C-terminus, [VH2-D]-[CH1]-[Fc1]; and The second light chain is, from the N-terminus to the C-terminus, [VL2-D]-[CL]. Here, VH1-I and VL1-I are the heavy chain variable region and light chain variable region of I0 in WO2021139758, respectively, and VH2-D and VL2-D are the heavy chain variable region and light chain variable region of D0 in WO2021139758, respectively. In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the DI bispecific antibody are shown in the table below.

[0215] [Table 6]

[0216] The binding activity of the DI-2 to DI-20 bispecific antibodies to their antigens was detected by the method in Test Example 4 of WO2021139758. The thermal stability of the antibodies was studied. Research method: The antibody was diluted to a concentration of 5 mg / mL with PBS, and its thermal stability was measured using a high-throughput differential scanning fluorometer (UNCHAINED, model number: Unit). The experimental results showed that the modified bispecific antibodies had no significant change in binding activity to the antigen, and compared with DI-2, DI-4 to DI-8, DI-10 to DI-16, and DI-20 had significantly improved Tm1 (℃) and Tonset (℃), indicating that the thermal stability of the bispecific antibodies was superior.

[0217] [Table 7-1] [Table 7-2] [Table 8]

[0218] A solution containing DI bispecific antibodies was prepared using a buffer solution of 10 mM acetic acid, pH 5.5, and 9% sucrose, and the solution was incubated in an incubator at 40°C for 4 weeks. After incubation, the antibody concentration was concentrated to the concentration at the start of incubation, and the precipitation state of the solution was observed. The experimental results showed that precipitation appeared in the solution of the DI-2 bispecific antibody group, and DI-3 to DI-7 have better stability than DI-2.

[0219] [Table 9]

[0220] 1.2 PL bispecific antibodies Anti-hPDL1 and hCTLA4 PL bispecific antibodies: PL-1 to PL-19 were constructed, which comprise the following first heavy chain, second heavy chain, first light chain and second light chain: First heavy chain: from the N-terminus to the C-terminus, [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1]; First light chain: from the N-terminus to the C-terminus, it is [VL1-P]-[Linker 2]-[Titin chain]; A second heavy chain is, from the N-terminus to the C-terminus, [VH2-L]-[CH1]-[Fc2]; and The second light chain is, from the N-terminus to the C-terminus, [VL2-L]-[CL]. Of these, VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733, respectively, and the amino acid sequences of VH2-L and VL2-L are as follows.

[0221] >VH2-L (SEQ ID NO: 191) [ka] >VL2-L (SEQ ID NO: 192) [ka]

[0222] In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the PL bispecific antibody are shown in the table below.

[0223] [Table 10]

[0224] The binding activity of the PL bispecific antibody was detected by referring to the ELISA method in Test Example 4 in WO2021139758, among which, hPDL1 and hCTLA4 antigens were purchased from Sino biology. The thermal stability of the antibody was studied. Method: The antibody was diluted with PBS to a concentration of 1.4-3 mg / mL, and its thermal stability was measured by a high-throughput differential scanning fluorometer (UNCHAINED, model number: Unit). The experimental results showed that the PL bispecific antibody still had good binding activity to the antigen, and compared with PL-1, PL-2 to PL-19 had significantly improved Tm1 (℃), Tagg 266 (℃), and Tonset (℃), and the thermal stability of the bispecific antibody was better.

[0225] [Table 11] [Table 12]

[0226] 1.3 HJ bispecific antibody Anti-hIL5 and hTSLP HJ bispecific antibodies: HJ-3 to HJ11 were constructed, which contain the following first heavy chain, second heavy chain, first light chain and second light chain: First heavy chain: from the N-terminus to the C-terminus, it is [VH1-H]-[Linker 1]-[Titin chain]-[Fc1]; First light chain: from the N-terminus to the C-terminus, [VL1-H]-[Linker 2]-[Obscurin chain]; A second heavy chain is, from the N-terminus to the C-terminus, [VH2-J]-[CH1]-[Fc2]; and The second light chain is, from the N-terminus to the C-terminus, [VL2-J]-[CL]; Here, VH1-H and VL1-H are the heavy chain variable region and light chain variable region of H0 in WO2021139758, respectively, and VH2-J and VL2-J are the heavy chain variable region and light chain variable region of J1 in WO2021139758, respectively. In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the HJ bispecific antibody are shown in the table below.

[0227] [Table 13]

[0228] The antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 of WO2021139758. The thermal stability of the antibody was studied as follows: a diluted solution of HJ bispecific antibody was prepared with a buffer solution of 10 mM acetic acid, pH 5.5, and 9% sucrose, and the bispecific antibody was concentrated by ultrafiltration concentration to obtain HJ bispecific antibody solutions of different concentrations (the concentrations of HJ bispecific antibody are shown in Table 13-2). The concentrated solutions were then incubated in a 40°C incubator, and the SEC purity of the samples was detected on day 0 (i.e., before the start of incubation at 40°C, D0), day 7 (day 7 of incubation at 40°C, D7), day 14 (day 14 of incubation at 40°C, D14), day 21 (day 21 of incubation at 40°C, D21) and day 28 (day 28 of incubation at 40°C, D28). After 28 days of incubation at 40°C, samples were immediately sampled to detect the CE-SDS purity of the samples. Experimental results show that the HJ bispecific antibodies constructed in the present disclosure have no significant change in antigen-binding activity, and compared with HJ-3, the HJ-5 to HJ-11 bispecific antibodies have better thermal stability.

[0229] [Table 14] [Table 15-1] [Table 15-2] Example 2. Construction and identification of cell lines expressing recombinant human GPRC5D and cynomolgus monkey GPRC5D

[0230] In order to screen for antibodies capable of binding relatively well to cell surface GPRC5D, the present disclosure constructed CHOK1-humanGPRC5D, HEK293-humanGPRC5D, CHOK1-cynoGPRC5D and HEK293-cynoGPRC5D cell lines expressing human GPRC5D or cynomolgus monkey GPRC5D. The full-length human GPRC5D or cynomolgus monkey GPRC5D gene was cloned into the mammalian cell expression vector pCDH, and the pVSV-G, pCMV-dR8.91 and pCDH-humanGPRC5D or pCDH-cynoGPRC5D plasmids were co-transfected into HEK293T cells (ATCC® CRL-11268) to package the virus. After 48 hours of transfection, the virus was collected and used to infect CHOK1 cells (ATCC® CCL-61) and HEK293 cells (ATCC® CRL-1573), respectively. After 72 hours of infection, monoclonal cells highly expressing human GPRC5D or cynomolgus monkey GPRC5D were obtained by flow cytometry selection. Example 3. Preparation of mouse anti-human GPRC5D monoclonal antibody

[0231] SJL mice were cross-immunized with CHOK1-humanGPRC5D and CHOK1-cynoGPRC5D cells. After seven immunizations, blood samples were taken to measure the antibody titers in the serum. Mice with high and stable antibody titers in the serum were selected for splenocyte fusion, and the fused hybridoma cells were seeded in a 96-well cell culture plate and cultured in an incubator at 37°C and 5% CO2. The cell culture supernatant was taken and detected by mirrorball and flow cytometry sorting techniques (FACS). The screened positive clones were amplified and frozen as seeds, and were subcloned 2 to 3 times until single-cell clones were obtained. Antibodies were further prepared and purified from the selected hybridoma clones by serum-free cell culture methods. The resulting hybridoma antibodies were subjected to FACS to detect binding to CHOK1-humanGPRC5D, CHOK1-cynoGPRC5D, and CHOK1 cells (see Test Example 1 of the present disclosure for the method), and hybridoma cell lines mAb1, mAb24, mAb30, and mab45, which had good human-monkey cross-binding activity and no nonspecific binding, were selected.

[0232] The sequences of monoclonal antibodies in hybridoma cell lines mAb1, mAb24, mAb30, and mab45 were cloned, and the process was as follows: hybridoma cells in logarithmic growth phase were harvested, and RNA was extracted with Trizol (Invitrogen, Cat#15596-018) and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification, and then sent to a sequencing company for sequencing. The amino acid sequences of the antibody variable regions corresponding to the obtained DNA sequences are as follows: Amino acid sequence of the mAb1 heavy chain variable region: [ka] Amino acid sequence of the mAb1 light chain variable region: [ka] Amino acid sequence of the mAb24 heavy chain variable region: [ka] Amino acid sequence of the mAb24 light chain variable region: [ka] Amino acid sequence of the mAb30 heavy chain variable region: [ka] Amino acid sequence of the mAb30 light chain variable region: [ka] Amino acid sequence of the mAb45 heavy chain variable region: [ka] Amino acid sequence of the mAb45 light chain variable region: [ka]

[0233] [Table 16-1] [Table 16-2] Example 4. Humanized design of anti-human GPRC5D monoclonal antibody

[0234] The humanization of mouse monoclonal antibodies was carried out according to the methods disclosed in many documents in this field. Briefly, based on the typical structure of the VH / VL CDR of the obtained mouse antibody, the homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) were searched for in the human germline database, the CDR region of the mouse antibody was grafted onto a human template, some of the residues of VL and VH were mutated, and the constant region of the mouse antibody was replaced with a human constant region to obtain the final humanized molecule.

[0235] 1. Humanization of mAb1 antibody

[0236] FR1, FR2, and FR3 of the humanized heavy chain variable region of mAb1 were IGHV3-21*01, and FR4 was IGHJ6*01, and FR1, FR2, and FR3 of the light chain variable region were IGKV2-29*02, and FR4 was IGKJ2*01. Optionally, amino acids at positions 49, 56, 57, and / or 93 of the humanized antibody heavy chain variable region were substituted, and / or amino acids at positions 2, 28, 29, and / or 45 of the humanized antibody light chain variable region were substituted.

[0237] [Table 17-1] [Table 17-2]

[0238] The sequences of the antibody variable regions are as follows (underlined CDRs, etc.): >hu1H1 (SEQ ID NO: 33): [ka] >hu1H2 (SEQ ID NO: 34): [ka] >hu1H3 (SEQ ID NO: 35): [ka] >hu1L1 (SEQ ID NO:36): [ka] >hu1L2 (SEQ ID NO:37): [ka] >hu1L3 (SEQ ID NO:38): [ka] >hu1L4 (SEQ ID NO:39): [ka] >hu1L5 (SEQ ID NO: 40): [ka] >hu1L6 (SEQ ID NO: 41): [ka]

[0239] The amino acid sequences of the replaced CDRs in the humanized antibody are shown in the table below: [Table 18]

[0240] 2. Humanization of mAb24 antibody FR1, FR2, and FR3 of the humanized heavy chain variable region of the mAb24 antibody are IGHV1-46*01, and FR4 is IGHJ6*01, and FR1, FR2, and FR3 of the light chain variable region are IGKV2-30*02, and FR4 is IGKJ4*01. Optionally, amino acids at positions 1, 24, 37, 69, 71, 73, and / or 93 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acids at positions 4, 28, 29, 36, and / or 37 in the light chain variable region of the humanized antibody were substituted.

[0241] [Table 19]

[0242] The sequences of the antibody variable regions are as follows: >hu24H1 (SEQ ID NO: 42): [ka] >hu24H2 (SEQ ID NO: 43): [ka] >hu24H3 (SEQ ID NO: 44): [ka] >hu24L1 (SEQ ID NO: 45): [ka] >hu24L2 (SEQ ID NO: 46): [ka] >hu24L3 (SEQ ID NO: 47): [ka] >hu24L4 (SEQ ID NO: 48): [ka] >hu24L5 (SEQ ID NO: 49): [ka] >hu24L6 (SEQ ID NO:50): [ka]

[0243] The amino acid sequences of the replaced CDRs in the humanized antibody are shown in the table below: [Table 20]

[0244] 3. Humanization of mAb30 antibody FR1, FR2, and FR3 of the humanized heavy chain variable region of the mAb30 antibody were IGHV1-3*01, and FR4 was IGHJ6*01, and FR1, FR2, and FR3 of the light chain variable region were IGKV1-12*01, and FR4 was IGKJ2*01. Optionally, amino acids at positions 1, 28, 43, 45, 54, 69, 71, 73, and / or 93 in the heavy chain variable region of the humanized antibody were substituted, and / or amino acids at positions 43, 46, and / or 85 in the light chain variable region of the humanized antibody were substituted.

[0245] [Table 21]

[0246] The sequences of the antibody variable regions are as follows: >hu30H1 (SEQ ID NO:51): [ka] >hu30H2 (SEQ ID NO:52): [ka] >hu30H3 (SEQ ID NO:53): [ka] >hu30H4 (SEQ ID NO:54): [ka] >hu30H5 (SEQ ID NO:55): [ka] >hu30L1 (SEQ ID NO:56): [ka] >hu30L2 (SEQ ID NO:57): [ka]

[0247] The amino acid sequences of the replaced CDRs in the humanized antibody are shown in the table below: [Table 22]

[0248] 4. Humanization of mAb45 antibody FR1, FR2, and FR3 of the humanized heavy chain variable region of the mAb45 antibody are IGHV3-7*01, and FR4 is IGHJ6*01, and FR1, FR2, and FR3 of the light chain variable region are IGKV2-28*01, and FR4 is IGKJ4*01. Optionally, amino acids at positions 30, 38, 49, 74, 75, 76, and / or 77 in the heavy chain variable region of the humanized antibody are substituted, and / or amino acids at positions 2, 28, 29, 45, 63, and / or 76 in the light chain variable region of the humanized antibody are substituted.

[0249] [Table 23-1] [Table 23-2]

[0250] The sequences of the antibody variable regions are as follows: >hu45H1 (SEQ ID NO:58): [ka] >hu45H2 (SEQ ID NO:59): [ka] >hu45H3 (SEQ ID NO: 60): [ka] >hu45H4 (SEQ ID NO:61): [ka] >hu45H5 (SEQ ID NO:62): [ka] >hu45H6 (SEQ ID NO: 63): [ka] >hu45H7 (SEQ ID NO: 64): [ka] >hu45H8 (SEQ ID NO: 65): [ka] >hu45H9 (SEQ ID NO:66): [ka] >hu45H10 (SEQ ID NO:67): [ka] >hu45H11 (SEQ ID NO:68): [ka] >hu45L1 (SEQ ID NO: 69): [ka] >hu45L2 (SEQ ID NO: 70): [ka] >hu45L3 (SEQ ID NO:71): [ka] >hu45L4 (SEQ ID NO:72): [ka] >hu45L5 (SEQ ID NO:73): [ka] >hu45L6 (SEQ ID NO:74): [ka] >hu45L7 (SEQ ID NO:75): [ka]

[0251] The amino acid sequences of the replaced CDRs in the humanized antibody are shown in the table below: [Table 24] Example 5. Construction of chimeric antibodies

[0252] The variable region sequences of mouse anti-GPRC5D antibodies mAb1, mAb24, mAb30, and mAb45 were combined with the light chain constant region shown in SEQ ID NO:96 and the heavy chain constant region shown in SEQ ID NO:215, respectively, to obtain chimeric antibodies Chi-1, Chi-24, Chi-30, and Chi-45. Exemplarily, Chi-1 represents a chimeric antibody comprising the mouse heavy chain variable region, light chain variable region, and the above constant region of mAb1, and the others are inferred in this manner. The sequences of the heavy chain constant region and light chain constant region of the chimeric antibodies are as follows: > Heavy chain constant region sequence (SEQ ID NO: 215): [ka] Heavy chain sequence of Chi-1 (SEQ ID NO:216): [ka] Light chain sequence of Chi-1 (SEQ ID NO:217): [ka] Heavy chain sequence of Chi-24 (SEQ ID NO:218): [ka] Light chain sequence of Chi-24 (SEQ ID NO:219): [ka] Heavy chain sequence of Chi-30 (SEQ ID NO:220): [ka] Light chain sequence of Chi-30 (SEQ ID NO:221): [ka] Heavy chain sequence of Chi-45 (SEQ ID NO:222): [ka] Light chain sequence of Chi-45 (SEQ ID NO:223): [ka] Example 6. Preparation and identification of anti-GPRC5D / CD3 bispecific antibodies

[0253] The CD3 binding molecule of the present disclosure may be derived from any suitable antibody. Particularly suitable antibodies are described, for example, in International Application No. PCT / CN2019 / 123548 (incorporated herein by reference in its entirety). Specifically, the embodiment of the present disclosure employs S107E.

[0254] [Table 25]

[0255] The sequence of the S107E variable region is as follows: >S107E-VH (SEQ ID NO: 82): [ka] >S107E-VL (SEQ ID NO: 83): [ka]

[0256] The humanized anti-GPRC5D antibody variable regions were combined with the variable regions of anti-CD3 antibody S107E, IgG1 (CH1), Obscurin, Titin, and the IgG1 mutant IgG1 (AA, L234A / L235A), to form several different forms of bispecific antibodies, Formula 2, Formula 2-0G4S, Formula 4, Formula 4-0G4S, and Formula 11, respectively.

[0257] Formula 2 (i.e., F2) is an asymmetric structural molecule having four chains, i.e., Chain 1: VH (anti-GPRC5D)-IgG1 (CH1)-IgG1Fc (Knob), Chain 2: VL(anti-GPRC5D)-CL, Chain 3 (OB(A1B0)-linker1-Hole): VH(S107E)-linker1-Obscurin(A1B0)-IgG1Fc(Hole), and Chain 4 (VL-linker2-Titin(A1B0)): VL(S107E)-linker2-Titin(A1B0), The schematic diagram is shown in FIG. 1A (OB stands for Obscurin).

[0258] Among them, >linker1 or linker2:GGGGS (SEQ ID NO:90) >OB(A1B0)-linker1-Hole (SEQ ID NO: 84): [ka]

[0259] >VL-linker2-Titin(A1B0) (SEQ ID NO:85): [ka]

[0260] Formula 2-0G4S (i.e., F2-0G4S) is an asymmetric structural molecule having four chains, i.e., Chain 1: VH (anti-GPRC5D)-IgG1 (CH1)-IgG1Fc (Knob), Chain 2: VL(anti-GPRC5D)-CL, Chain 3 (OB(A0B2)-Hole): VH(S107E)-OB(A0B2)-IgG1Fc(Hole), and Chain 4 (VL-Titin(A0B2)): VL(S107E)-Titin(A0B2), The schematic diagram is shown in FIG. 1B (OB stands for Obscurin).

[0261] Among them, >OB(A0B2)-Hole (SEQ ID NO:86): [ka]

[0262] >VL-Titin (A0B2) (SEQ ID NO: 87): [ka]

[0263] Formula 4 (i.e., F4) is an asymmetric structural molecule having four chains, i.e., Chain 1 (OB(A1B0)-linker3-Knob):VH(S107E)-linker3-OB(A1B0)-IgG1Fc(Knob), Chain 2 (VL-linker4-Titin(A1B0)): VL(S107E)-linker4-Titin(A1B0), Chain 3: VH(anti-GPRC5D)-IgG1(CH1)-IgG1Fc(Hole), and Chain 4: VL(anti-GPRC5D)-CL, The schematic diagram is shown in FIG. 1(C) (OB stands for Obscurin).

[0264] >linker3 or linker4:GGGGS (SEQ ID NO:90) >OB(A1B0)-linker3-Knob (SEQ ID NO: 88): [ka]

[0265] >VL-linker4-Titin (A1B0) (SEQ ID NO: 85): [ka]

[0266] Formula 4-0G4S (i.e., F4-0G4S) is an asymmetric structural molecule having four chains, i.e., Chain 1 (OB(A0B2)-Knob): VH(S107E)-OB(A0B2)-IgG1Fc(Knob), Chain 2 (VL-Titin(A0B2)): VL(S107E)-Titin(A0B2), Chain 3: VH(anti-GPRC5D)-IgG1(CH1)-IgG1Fc(Hole), and Chain 4: VL(anti-GPRC5D)-CL, The schematic diagram is shown in FIG. 1(D) (OB stands for Obscurin).

[0267] >Ob(A0B2)-Knob (SEQ ID NO:89): [ka]

[0268] >VL-Titin (A0B2) (SEQ ID NO: 87): [ka]

[0269] Among them, >IgG1 (CH1) (SEQ ID NO: 91): [ka] >IgG1Fc(Knob) (SEQ ID NO:92): [ka] >IgG1Fc(Hole)(SEQ ID NO:93): [ka] Formula 11 (ie, F11) is a symmetric molecule, containing two identical heavy chains (chain 1) and two identical light chains (chain 2).

[0270] Chain 1 (heavy chain): VH(anti-GPRC5D)-IgG1(CH1)-VH(S107E)-linker5-VL(S107E)-linker6- IgG1(AA)Fc, Chain 2 (light chain): VL(anti-GPRC5D)-CL, The schematic diagram is shown in FIG. 1E.

[0271] Among them, >linker5:GGGGSGGGGSGGGGS (SEQ ID NO:94) >linker6:GGG (sequence number 95) >IgG1 (CH1) (SEQ ID NO: 91): [ka] >CL (SEQ ID NO: 96): [ka] >IgG1(AA) (SEQ ID NO: 97): [ka]

[0272] Based on the humanized amino acid sequences of mAb1, mAb24, mAb30, and mAb45, the following bispecific antibodies were constructed.

[0273] [Table 26-1] [Table 26-2] [Table 26-3]

[0274] Note: In Table 24, F11-1 indicates that the molecule uses the variable region of mAb1 and its humanized antibody as the GPRC5D binding domain and adopts formula 11 as the molecular structure, F2-1 indicates that the molecule uses the variable region of mAb1 and its humanized antibody as the GPRC5D binding domain and adopts formula 2 as the molecular structure, and the others are inferred similarly.

[0275] The specific amino acid sequence is as follows: Chain 1 of F11-1, F11-2, F11-3 (>hu1H2-F11-1 (SEQ ID NO:98)): [ka] Chain 1 of F11-4, F11-5, F11-6 (>hu1H3-F11-1 (SEQ ID NO:99)): [ka] Chain 2 of F11-1, F11-4 (>hu1L1-CL (SEQ ID NO: 100)): [ka] Chain 2 of F11-2, F11-5 (>hu1L3-CL (SEQ ID NO: 101)): [ka] Chain 2 of F11-3, F11-6 (>hu1L4-CL (SEQ ID NO: 102)): [ka] Chain 1 of F11-7, F11-8, F11-9 (>hu24H2-F11-1 (SEQ ID NO: 103)): [ka] Chain 1 of F11-10, F11-11, F11-12 (>hu24H3-F11-1 (SEQ ID NO: 104)): [ka] Chain 2 of F11-7, F11-10 (>hu24L2-CL (SEQ ID NO: 105)): [ka] F11-8, chain 2 of F11-11 (>hu24L5-CL (SEQ ID NO: 106)): [ka] Chain 2 of F11-9, F11-12 (>hu24L6-CL (SEQ ID NO: 107)): [ka] Chain 1 of F11-13, F11-15 (>hu30H4-F11-1 (SEQ ID NO: 108)): [ka] Chain 1 of F11-14, F11-16 (>hu30H5-F11-1 (SEQ ID NO: 109)): [ka] Chain 2 of F11-13, F11-14 (>hu30L1-CL (SEQ ID NO:110)): [ka] Chain 2 of F11-15, F11-16 (>hu30L2-CL (SEQ ID NO:111)): [ka] Chain 3 of F4-1 (>hu45H4-CH1-Hole (SEQ ID NO:112)): [ka] Chain 3 of F4-2 (>hu45H5-CH1-Hole (SEQ ID NO:113)): [ka] Strand 3 of F4-3 (>hu45H7-CH1-Hole (SEQ ID NO:114)): [ka] Chain 3 of F4-4 (>hu45H8-CH1-Hole (SEQ ID NO:115)): [ka] Strand 3 of F4-5 (>hu45H10-CH1-Hole (SEQ ID NO:116)): [ka] Chain 4 of F4-1, F4-2, F4-3, F4-4, F4-5 (>hu45L7-CL (SEQ ID NO: 117)): [ka] Chain 1 of F2-0G4S-1 (>hu1H3-CH1-Knob (SEQ ID NO:118)): [ka] Chain 3 of F4-8 (>hu1H3-CH1-Hole (SEQ ID NO:119)): [ka] Chain 1 of F2-2 (>hu24H3-CH1-Knob (SEQ ID NO: 120)): [ka] Chain 3 of F4-6 (>hu24H3-CH1-Hole (SEQ ID NO: 121)): [ka] Chain 1 of F2-3 (>hu30H5-CH1-Knob (SEQ ID NO: 122)): [ka] Chain 3 of F4-7 (>hu30H5-CH1-Hole (SEQ ID NO: 123)): [ka] Chain 1 of F2-4 (>hu45H10-CH1-Knob (SEQ ID NO:124)): [ka] Chain 1 of F11-17 (>Chi-1-F11-1) (SEQ ID NO:224): [ka] TIFF2025508160000146.tif30162 Strand 2 of F11-17 (>Chi-1VL-CL) (SEQ ID NO:217): [ka] Chain 1 of F11-18 (>Chi-24-F11-1) (SEQ ID NO:225): [ka] Chain 2 of F11-18 (>Chi-24VL-CL) (SEQ ID NO:219): [ka] Chain 1 of F11-19 (>Chi-30-F11-1) (SEQ ID NO:226): [ka] Chain 2 of F11-19 (>Chi-30VL-CL) (SEQ ID NO:221): [ka] Strand 3 of F4-9 (>Chi-45-CHI-Hole) (SEQ ID NO:227): [ka] Chain 4 of F4-9 (>Chi-45VL-CL) (SEQ ID NO:223): [ka]

[0276] The positive control molecule used in this disclosure is JNJ-64407564. The sequence is as follows (from WO2018017786): > Heavy chain (anti-GPRC5D) (SEQ ID NO: 125): [ka] >Light chain (anti-GPRC5D) (SEQ ID NO: 126): [ka] > Heavy chain (anti-CD3E) (SEQ ID NO: 127): [ka] >Light chain (anti-CD3E) (SEQ ID NO: 128): [ka] > Heavy chain variable region of humanized anti-GPRC5D antibody in JNJ-64407564 (SEQ ID NO: 228): [ka] >Light chain variable region of humanized anti-GPRC5D antibody in JNJ-64407564 (SEQ ID NO: 229): [ka] Heavy chain of JNJ7564-huIgG1 (SEQ ID NO: 230): [ka] >Light chain of JNJ7564-huIgG1 (SEQ ID NO: 231): [ka] Test Example Test Example 1. Measurement of affinity of GPRC5D / CD3 bispecific antibody to GPRC5D by FACS

[0277] In order to test the binding ability of the GPRC5D / CD3 bispecific antibody according to the present disclosure to GPRC5D, this test example was carried out by flow cytometry using a human multiple myeloma cell line MM.1R (ATCC 61161) expressing GPRC5D. (登録商標) The binding ability of the GPRC5D / CD3 bispecific antibody to the stable transformed cell line CHOK1-cynoGPRC5D expressing cynomolgus monkey GPRC5D was detected. Specifically, the above cells were cultured in a complete medium containing 10% FBS, placed in an incubator at 37°C and 5% CO2 for 2 days, and 1 × 10 5 Cells were added to the cell plate at 100μL / well, centrifuged at 300g for 5 min, and washed once with 1% BSA. Antibody concentrations were serially diluted to 8 concentrations, added to the cell plate at 100μL / well, incubated at 4℃ for 1 h, washed once with 1% BSA, and 100μL of FITC-Goat Anti-human IgG Fc fluorescent secondary antibody dilution (1:400) was added to each well and incubated at 4℃ for 1 h. The plate was washed three times with 1% BSA, and the plate was read by adding 100μL of PBS to each well. EC binding to the corresponding cells for each antibody was calculated. 50 (nM) is as shown in the table below.

[0278] [Table 27]

[0279] [Table 28] [Table 29-1] [Table 29-2] [Table 30]

[0280] [Table 31]

[0281] [Table 32-1] [Table 32-2]

[0282] As a result, various GPRC5D monoclonal antibodies or GPRC5D- / CD3 bispecific antibodies constructed by the GPRC5D antibodies screened in the present disclosure have been shown to have excellent binding ability to membrane surface GPRC5D and relatively good cross-binding activity to CynoGPRC5D. JNJ-64407564 has a weaker binding ability to humanGPRC5D than the antibodies disclosed in the present disclosure, mainly due to its low binding response value, and has very weak affinity to cynoGPRC5D, with almost no binding detected. Test Example 2. Measurement of the binding epitope of GPRC5D / CD3 bispecific antibody to GPRC5D by ELISA

[0283] To determine the binding epitope of the GPRC5D / CD3 bispecific antibody of the present disclosure to GPRC5D, four extracellular domain peptides of human GPRC5D, including the amino-terminal peptide (N-terminal peptide), E2 peptide, E3 peptide, and E4 peptide, were synthesized (synthesized by GenScript). The peptides were biotinylated and formed into two forms, intramolecular disulfide bond-deficient and intramolecular disulfide bond-containing, and the sequences are shown in Table 26. An Elisa plate (corning 3590) was coated with steptavidin (sigma, SA101), incubated at 37°C for 2 hours, and then washed. Non-specific binding sites were blocked with PBS containing 5% skim milk, incubated at 37°C for 2 hours, and then washed. 100 μL of peptide diluted with PBS containing 1% BSA was added to each well. The plate was incubated at 37°C for 1 hour, and then washed. Then, 100 μL of GPRC5D / CD3 antibody diluted to 0.003 nM to 300 nM with PBS containing 1% BSA was added to each well. The plate was incubated at 37°C for 1 hour, and then washed. Goat anti-human IgG(H+L)-HRP (Jackson ImmunoResearch, 209-035-088) was added, and the plate was left to stand at 37°C for 1 hour, after which the plate was washed, and 100 μL of TMB (KPL, 5120-0077) was added to each well. Color development was allowed to continue for 5 minutes, and then 1 M H2SO4 was added to stop the development. 450 The specific results are shown in Table 27.

[0284] [Table 33-1] [Table 33-2] [Table 34]

[0285] The results show that the GPRC5D / CD3 bispecific antibodies F2-0G4S-1 and F4-0G4S-1 of the present disclosure bind to the amino-terminal peptide sequence of human GPRC5D, regardless of whether the peptide forms an intramolecular disulfide bond. At the same time, all detection antibodies did not bind to the E2 peptide, E3 peptide, or E4 peptide. These results show that the binding epitopes of the GPRC5D / CD3 bispecific antibodies F2-0G4S-1 and F4-0G4S-1 of the present disclosure to GPRC5D are located in the amino-terminal region of human GPRC5D. Test Example 3. Measurement of cross-binding of GPRC5D / CD3 bispecific antibodies to GPCRA and GPCRB family members by FACS

[0286] To test whether the GPRC5D / CD3 bispecific antibody of the present disclosure cross-links with GPCRA and GPCRB family members, this test example used a flow cytometry method to detect the binding ability of the GPRC5D / CD3 bispecific antibody to CHOK1-human CCR8 cells expressing human CCR8 (GPCR A family) (see construction in Example 2) and CHOK1-human GIPR cells expressing human GIPR (GPCR B family) (see construction in Example 2). Specifically, the above cell culture was cultured in DMEF / 12K medium containing 10% FBS, placed in an incubator at 37°C and 5% CO2 for 2 days, and 1×10 5 Cells were added to the cell plate at 100 cells / well, centrifuged at 300g for 5 min, and washed once with 1% BSA. Antibody concentrations were serially diluted to 8 concentrations, added to the cell plate at 100 μL / well, incubated at 4°C for 1 h, washed once with 1% BSA, and 100 μL of FITC-Goat Anti-human IgG Fc fluorescent secondary antibody dilution (1:400) was added to each well and incubated at 4°C for 1 h. The plate was washed 3 times with 1% BSA, and 100 μL of PBS was added to each well to read the plate.

[0287] >Human CCR8 antigen (SEQ ID NO: 211): [ka] >Human GIPR antigen (SEQ ID NO: 212): [ka]

[0288] As a result, none of the GPRC5D / CD3 bispecific antibodies F2-1, F2-0G4S-1, F4-6 and F4-0G4S-1 of the present disclosure bound to human CCR8 or human GIPR, demonstrating their excellent specificity. Test Example 4. In vitro cytotoxic activity of bispecific antibodies according to the present disclosure

[0289] This test example investigated the tumor cell killing activity of the bispecific antibody according to the present disclosure, which serves as a T cell adaptor molecule. The target-specific cytotoxic activity of the bispecific antibody according to the present disclosure was detected using the cell line H929 expressing GPRC5D and Daudi not expressing GPRC5D as target cells. Fresh PBMCs (purchased from Milestone Biotechnologies) were centrifuged at 300g for 10 minutes, the supernatant was removed, and the cells were resuspended in 1640+10% FBS (solution A) containing 200U of IL2 to a cell suspension of 2E6 / mL, and cultured overnight in a culture flask. The PBMCs activated overnight were collected and centrifuged, then resuspended in phenol red-free 1640+4% FBS, centrifuged again, resuspended and counted, the cell number was adjusted to 6.25E5 cells / mL, and 60 μL was added to each well (the PBMC cell number in each well was 37,500). Target cells NCI-H929 (ATCC, CRL-9068) were collected and centrifuged at 1000 rpm for 3 min, the cell number was adjusted to 1.25E5 cells / mL, and 60 μL was added to each well so that the E:T ratio was 5:1 (the number of target cells in each well was 7500). The antibodies were diluted in phenol red-free complete medium with an initial concentration of 500 nM (5× final concentration), diluted 10-fold in nine gradients, and 30 μL was added to each well. The cells were then cultured in a 37°C, 5% CO2 incubator for 48 h. Before detection, 10 μL of medium was sucked out from two of the wells containing only target cells, and 10 μL of Lysis Solution (10×) was added. After 45 min of lysis, the culture plate was removed and centrifuged at 1000 rpm for 3 minutes, 50 μL of the supernatant was sucked out and placed in a new 96-well plate (#3590), and 50 μL of dissolved CytoTox 96® Reagent was added in a 1:1 ratio. After incubation at room temperature for 0.5 h, 50 μL of stop solution was added, and the absorbance (490 nm) was detected by FlexStation 3 (Molecular Devices). The specific results are shown in the table below.

[0290] [Table 35]

[0291] As a result, the GPRC5D / CD3 bispecific antibody of the present disclosure has stronger NCI-H929 cytotoxic activity than JNJ-64407564, and the cytotoxic activity of the GPRC5D / CD3 bispecific antibodies F2-2, F2-3, F4-6, F4-7, F2-0G4S-1 and F4-0G4S-1 of the present disclosure is specific to the GPRC5D target, and no killing activity was detected in the Daudi cell line, which does not express GPRC5D, suggesting that the GPRC5D / CD3 bispecific antibody of the present disclosure has better safety. Test Example 5. Cytokine release levels of GPRC5D / CD3 bispecific antibodies

[0292] CD3 T cell adaptor molecules will cause a cytokine storm. Therefore, when developing CD3 T cell adaptor molecules, it is necessary to maintain low levels of cytokines, especially IL6, a factor that is unrelated to drug efficacy but causes side effects. In this test example, the cytokine IFNγ and IL6 release levels of the bispecific antibody of the present disclosure were studied. Fresh PBMCs (purchased from Milestone Biotechnologies) were centrifuged at 300g for 10 minutes, the supernatant was removed, and the cells were resuspended in 1640+10% FBS (solution A) containing 200U of IL2 to a cell suspension of 2E6 / mL, and cultured in a culture flask overnight. The overnight activated PBMCs were collected and centrifuged, then resuspended in phenol red-free 1640+4% FBS, centrifuged again, resuspended and counted, the cell number was adjusted to 6.25E5 cells / mL, and 60 μL was added to each well (the PBMC cell number in each well is 37500). Target cells NCI-H929 (ATCC, CRL-9068) or RPMI-8226 (ATCC, CRM-CCL-155) were collected and centrifuged at 1000 rpm for 3 min, the cell number was adjusted to 1.25E5 cells / mL, and 60 μL was added to each well so that the E:T ratio was 5:1 (the number of target cells in each well was 7500). The antibodies were diluted in phenol red-free complete medium, with an initial concentration of 500 nM (5× final concentration), and 10-fold dilutions in nine gradients were added to each well, with 30 μL added. The cells were then cultured in a 37°C, 5% CO2 incubator for 48 h before use.

[0293] IL6 was detected by ELISA. The above cell samples were centrifuged at 1000 rpm for 3 minutes, 50 μL of cell supernatant was collected and diluted 6 times with the universal diluent for specimens, and the IL6 standard was also diluted 6 times. The diluted samples or standards of different concentrations were added to the detection plate (100 μL / well), the reaction wells were sealed with plate sealing adhesive paper, incubated at 37 °C for 90 minutes, and then the plate was washed 5 times, the biotinylated antibody working solution was added (100 μL / well), the reaction wells were sealed with new plate sealing adhesive paper, incubated at 37 °C for 60 minutes, and then the plate was washed 5 times, the enzyme complex working solution was added (100 μL / well), the reaction wells were sealed with new plate sealing adhesive paper, incubated at 37 °C for 30 minutes, and then the plate was washed 5 times, the color-developing substrate (TMB) was added at 100 μL / well, and incubated at 37 °C for 8 minutes in the dark, and the reaction stop solution was added at 100 μL / well. After uniform mixing, the OD was immediately measured. 450 The value was measured (within 3 minutes).

[0294] HTRF method was used to detect IFNγ. The above cell samples were centrifuged at 1000 rpm for 3 minutes, 50 μL of cell supernatant was collected, the detection reagent kit was equilibrated to room temperature, and the two detection antibodies in the reagent kit were diluted (20 times diluted) with detection buffer. 16 μL of the 20 times diluted sample and IFNγ standard were taken and placed in a 384-well plate, 4 μL of the diluted corresponding detection antibody was added, the sealing material was attached, shaken to mix uniformly, centrifuged at 1000 rpm for 1 minute, incubated overnight at room temperature, centrifuged at 1000 rpm for 1 minute, the sealing material was removed, and the absorbance values ​​at 665 nm and 620 nm were read by PHERAstar multifunction plate reader, and the data was processed and analyzed by Graphpad Prism 5. The specific results are shown in the table below.

[0295] [Table 36]

[0296] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41]

[0297] As a result, it was shown that the GPRC5D / CD3 bispecific antibody of the present disclosure released lower levels of IL6 and IFNγ than JNJ-64407564, and in particular, IL6 was hardly detected. It is suggested that the GPRC5D / CD3 bispecific antibody of the present disclosure has better safety. Biological evaluation of in vivo activity

[0298] Test Example 6. Efficacy of GPRC5D / CD3 bispecific antibody in NCIH929 subcutaneously transplanted tumor model

[0299] The present disclosure used a human myeloma NCI-H929 cell human PBMC NOG mouse xenograft tumor model to evaluate GPRC5D / CD3 bispecific antibodies for anti-tumor activity.

[0300] Female NOG mice, 8–10 weeks old, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0301] Human multiple myeloma cell line NCI-H929 was purchased from Shanghai Mingjing Biological Technology Co., Ltd.

[0302] NCI-H929 cells were cultured in RPMI-1640 medium containing 10% FBS and 0.05 mM 2-mercaptoethanol in a 5% CO2, 37°C saturated humidity incubator. NCI-H929 cells in logarithmic growth phase were harvested and resuspended in RPMI-1640 medium containing 50% Matrigel to a cell concentration of 2 × 10 7 Under sterile conditions, 0.1 mL of the cell suspension was inoculated subcutaneously into the right dorsum of each mouse, resulting in an inoculum concentration of 2 × 10 6 The total volume of the cultured hPBMC was 0.1 mL / mouse. One day after tumor cell inoculation, the hPBMCs frozen in resuscitated liquid nitrogen were cultured in PRMI-1640 medium containing 10% HIFBS (FBS, inactivated at 56°C for 30 min) and incubated in a 37°C incubator containing 5% CO2 for 6 h. The incubated hPBMCs were collected and resuspended in PBS buffer to a cell concentration of 2.5 x 10 7 Under sterile conditions, 0.2 mL of the cell suspension was intraperitoneally injected into the mouse, with the injection concentration adjusted to 5 × 10 6 The concentration was 0.2 mL / mouse. The mean tumor volume was 100 mm 3 When the tumor volume reached 100%, the animals were randomly divided into groups according to tumor volume so that the tumor volume difference between each group was less than 10% of the mean value. The day of grouping was marked as Day 0, and administration began according to the weight of the animals. Administration was started immediately after grouping, intraperitoneal administration once a week, and two consecutive administrations (IP, qW x 2), and the tumors were weighed and recorded.

[0303] Tumor inhibition rate (%) = 1-T / C (%). T / C(%)=(T-T0) / (C-C0)×100%. Tumor volume (V) = 1 / 2 × a × b 2 where a and b indicate length and width, respectively.

[0304] Among them, T and C are the tumor volumes of the treatment group and the control group at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0305] The specific results are shown in Tables 30-1, 30-2, 30-3 and FIG. 2, where the tumor inhibition rates calculated in the tables are all tumor inhibition rates on the final day.

[0306] [Table 42] [Table 43] [Table 44]

[0307] Experimental results show that the disclosed GPRC5D / CD3 bispecific antibodies F2-1, F2-4, F4-6, F4-7, F4-8, F2-0G4S-1 and F4-0G4S-1 have higher in vivo tumor growth inhibitory activity than the corresponding dose of JNJ-64407564 under the above dosage conditions. No drug-related animal deaths or other obvious drug-related toxicities or side effects were observed during the experiment.

[0308] It is to be clearly understood that while the foregoing invention has been described in detail by way of illustration and example, these descriptions and examples are not to be construed as limiting the scope of this disclosure, and the disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. An antigen-binding molecule that specifically binds to GPRC5D and CD3, comprising at least one antigen-binding module that specifically binds to GPRC5D and at least one antigen-binding module that specifically binds to CD3, wherein the antigen-binding module that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, and the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein (i) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, 18, 135, 136, or 138, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, 10 or 133, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, 12, 129, 131 or 132, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; or (iii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 140, 22, or 139, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or (iv) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 144, 30, 141, 142, or 143, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; Preferably, (i) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; Antigen-binding molecules.

2. The antigen binding module that specifically binds to GPRC5D comprises: (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, 3, 42 or 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, 1, 33 or 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, 5, 51, 52, 53, or 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56, 6, or 57; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, 7, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 68, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75, 8, 69, 70, 71, 72, 73 or 74; Preferably, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:3, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:4; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:1, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:2; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:54, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:56; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:54, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:57; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:55, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:57; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:5, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:6; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:61, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:62, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:64, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:65, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:7, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:8; or More preferably, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; The antigen-binding molecule of claim 1.

3. the antigen-binding module that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in the CD3-VH comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 82, respectively, and CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in the CD3-VL comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 83, respectively; Preferably, The CD3-VH has a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 76, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 78, and the CD3-VL has a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 79, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 81, More preferably, The CD3-VH comprises the amino acid sequence of SEQ ID NO: 82, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 83; The antigen-binding molecule of claim 1 or 2.

4. The antigen-binding molecule further comprises an Fc region, and the Fc region is preferably an IgG Fc region, and more preferably an IgG １ More preferably, the Fc Region comprises one or more amino acid substitutions capable of reducing binding of the Fc Region to an Fcγ receptor, Most preferably, the Fc region is human IgG １ Fc region and the amino acids at positions 234 and 235 are A, numbered according to the EU index; The antigen-binding molecule according to any one of claims 1 to 3.

5. the Fc region comprises a first subunit, Fc1, and a second subunit, Fc2, capable of associating with each other, and the Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region; Preferably, the Fc1 has a knob structure based on knob-in-hole technology, and the Fc2 has a hole structure based on knob-in-hole technology, or the Fc1 has a hole structure based on knob-in-hole technology, and the Fc2 has a knob structure based on knob-in-hole technology; More preferably, said Fc1 has an amino acid at position 366 that is W, and said Fc2 has an amino acid at position 366 that is S, an amino acid at position 368 that is A, and an amino acid at position 407 that is V, numbered according to the EU index. The antigen-binding molecule of claim 4.

6. the antigen-binding module that specifically binds to GPRC5D is a Fab, and the antigen-binding module that specifically binds to CD3 is a substituted Fab, or the antigen-binding module that specifically binds to GPRC5D is a substituted Fab and the antigen-binding module that specifically binds to CD3 is a Fab, and the substituted Fab has original CH1 and CL replaced with an Obscurin chain and a Titin chain, respectively, or has original CH1 and CL replaced with a Titin chain and an Obscurin chain, respectively; Preferably, The Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 145-163, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-204; More preferably, The Titin chain comprises the amino acid sequence of SEQ ID NO: 163 or 161, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 198 or 199; Most preferably, the Titin chain comprises the amino acid sequence of SEQ ID NO: 163 and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 198; or The Titin chain comprises the amino acid sequence of SEQ ID NO: 161, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 199; The antigen-binding molecule according to any one of claims 1 to 5.

7. At least one antigen-binding module that specifically binds to GPRC5D and at least one antigen-binding module that specifically binds to CD3 are linked to the N-terminus of an Fc1 or Fc2 subunit directly or via a linker at the C-terminus of the CH1 or Obscurin chain, and are associated as a heterodimer by disulfide bonds at the N-terminus of the Fc1 and Fc2 subunits. The antigen-binding molecule of claim 5 or 6.

8. The antigen-binding molecule comprises one first chain having a structure represented by formula (a), one second chain having a structure represented by formula (b), one third chain having a structure represented by formula (c), and one fourth chain having a structure represented by formula (d); (a) [GPRC5D-VH]-[CH1]-[Fc1], (b) [GPRC5D-VL]-CL, (c) [CD3-VH]-[Obscurin chain]-[Fc2], (d) [CD3-VL]-[Titin chain], The structures represented by formulae (a), (b), (c) and (d) are arranged from the N-terminus to the C-terminus, Preferably, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 118, one second chain comprising the amino acid sequence of SEQ ID NO: 102, one third chain comprising the amino acid sequence of SEQ ID NO: 86, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

87. The antigen-binding molecule according to any one of claims 1 to 7.

9. The antigen-binding molecule has one first chain having a structure represented by formula (e), one second chain having a structure represented by formula (d), one third chain having a structure represented by formula (f), and one fourth chain having a structure represented by formula (b); (e) [CD3-VH]-[Obscurin chain]-[Fc1]; (d) [CD3-VL]-[Titin chain], (f) [GPRC5D-VH]-[CH1]-[Fc2], (b) [GPRC5D-VL]-CL, The structures represented by formulae (e), (d), (f) and (b) are arranged from the N-terminus to the C-terminus, Preferably, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 89, one second chain comprising the amino acid sequence of SEQ ID NO: 87, one third chain comprising the amino acid sequence of SEQ ID NO: 121, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

106. The antigen-binding molecule described in claim 7.

10. The antigen-binding molecule has one first chain having a structure represented by formula (a), one second chain having a structure represented by formula (b), one third chain having a structure represented by formula (g), and one fourth chain having a structure represented by formula (h); (a) [GPRC5D-VH]-[CH1]-[Fc1], (b) [GPRC5D-VL]-CL, (g) [CD3-VH]-linker1-[Obscurin chain]-[Fc2]; (h) [CD3-VL]-linker2-[Titin chain]; The structures represented by formulae (a), (b), (g) and (h) are arranged from the N-terminus to the C-terminus, and the linker 1 and the linker 2 are the same or different peptide linkers; Preferably, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 118, one second chain comprising the amino acid sequence of SEQ ID NO: 102, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 120, one second chain comprising the amino acid sequence of SEQ ID NO: 106, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 122, one second chain comprising the amino acid sequence of SEQ ID NO: 110, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 85; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 124, one second chain comprising the amino acid sequence of SEQ ID NO: 117, one third chain comprising the amino acid sequence of SEQ ID NO: 84, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

85. The antigen-binding molecule described in claim 7.

11. The antigen-binding molecule has one first chain having a structure represented by formula (i), one second chain having a structure represented by formula (j), one third chain having a structure represented by formula (f), and one fourth chain having a structure represented by formula (b); (i) [CD3-VH]-linker3-[Obscurin chain]-[Fc1]; (j) [CD3-VL]-linker4-[Titin chain]; (f) [GPRC5D-VH]-[CH1]-[Fc2], (b) [GPRC5D-VL]-CL, The structures represented by formulae (i), (j), (f) and (b) are arranged from the N-terminus to the C-terminus, and the linker 3 and the linker 4 are the same or different peptide linkers; Preferably, The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 116, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 117; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 121, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 106; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 123, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 110; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 119, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 102; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 115, and one fourth chain comprising the amino acid sequence of SEQ ID NO: 117; or The antigen-binding molecule has one first chain comprising the amino acid sequence of SEQ ID NO: 88, one second chain comprising the amino acid sequence of SEQ ID NO: 85, one third chain comprising the amino acid sequence of SEQ ID NO: 227, and one fourth chain comprising the amino acid sequence of SEQ ID NO:

223. The antigen-binding molecule described in claim 7.

12. the antigen-binding molecule comprises two antigen-binding modules that specifically bind to GPRC5D and two antigen-binding modules that specifically bind to CD3, the antigen-binding module that specifically binds to GPRC5D is a Fab, and the antigen-binding module that specifically binds to CD3 is an scFv; Preferably, The antigen-binding molecule has two first chains having a structure represented by formula (k) and two second chains having a structure represented by formula (b), (k) [GPRC5D-VH]-[CH1]-[CD3-VH]-linker5-[CD3-VL]-linker6-[one subunit of Fc]; (b) [GPRC5D-VL]-CL, The structures represented by formulae (k) and (b) are arranged from the N-terminus to the C-terminus, and the linker 5 and the linker 6 are the same or different peptide linkers; More preferably, The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 98 and two second chains comprising the amino acid sequence of SEQ ID NO: 100; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 98 and two second chains comprising the amino acid sequence of SEQ ID NO: 101; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 98 and two second chains comprising the amino acid sequence of SEQ ID NO: 102; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 99 and two second chains comprising the amino acid sequence of SEQ ID NO: 100; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 99 and two second chains comprising the amino acid sequence of SEQ ID NO: 101; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 99 and two second chains comprising the amino acid sequence of SEQ ID NO: 102; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 103 and two second chains comprising the amino acid sequence of SEQ ID NO: 105; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 103 and two second chains comprising the amino acid sequence of SEQ ID NO: 106; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 103 and two second chains comprising the amino acid sequence of SEQ ID NO: 107; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 104 and two second chains comprising the amino acid sequence of SEQ ID NO: 105; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 104 and two second chains comprising the amino acid sequence of SEQ ID NO: 106; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 104 and two second chains comprising the amino acid sequence of SEQ ID NO: 107; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 108 and two second chains comprising the amino acid sequence of SEQ ID NO: 110; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 109 and two second chains comprising the amino acid sequence of SEQ ID NO: 110; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 108 and two second chains comprising the amino acid sequence of SEQ ID NO: 111; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 109 and two second chains comprising the amino acid sequence of SEQ ID NO: 111; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 224 and two second chains comprising the amino acid sequence of SEQ ID NO: 217; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 225 and two second chains comprising the amino acid sequence of SEQ ID NO: 219; or The antigen-binding molecule has two first chains comprising the amino acid sequence of SEQ ID NO: 226 and two second chains comprising the amino acid sequence of SEQ ID NO:

221. The antigen-binding molecule of claim 5.

13. An isolated antibody capable of specifically binding to GPRC5D, said antibody comprising a heavy chain variable domain GPRC5D-VH and a light chain variable domain GPRC5D-VL, wherein: (i) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, 18, 135, 136, or 138, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, 10 or 133, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, 12, 129, 131 or 132, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; or (iii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 21, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 140, 22, or 139, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or (iv) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 29, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 144, 30, 141, 142, or 143, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; Preferably, (i) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 17, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 137, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (ii) the GPRC5D-VH has a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 134, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, and the GPRC5D-VL has a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 130, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; Isolated antibodies.

14. (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, 3, 42 or 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49, 4, 45, 46, 47, 48 or 50; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, 1, 33 or 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39, 2, 36, 37, 38, 40 or 41; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, 5, 51, 52, 53, or 54, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56, 6, or 57; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, 7, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 68, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75, 8, 69, 70, 71, 72, 73 or 74; Preferably, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 43, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 50; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:3, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:4; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 38; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:1, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:2; or (iii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 55, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:54, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:56; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:54, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:57; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:55, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:57; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:5, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:6; or (iv) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 67, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:61, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:62, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:64, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or said GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:65, and said GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:75; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO:7, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO:8; or More preferably, (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 44, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 49; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 35, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39; 14. An isolated antibody according to claim 13.

15. It is a bispecific antibody, Preferably, the bispecific antibody specifically binds to GPRC5D and CD3.

15. An isolated antibody according to claim 13 or 14.

16. does not bind to E2 peptide, E3 peptide, or E4 peptide, wherein the E2 peptide comprises the amino acid sequence of SEQ ID NO: 206, the E3 peptide comprises the amino acid sequence of SEQ ID NO: 207, and the E4 peptide comprises the amino acid sequence of SEQ ID NO: 208; Preferably, it binds to the amino terminal region of human GPRC5D, wherein the amino terminal region of human GPRC5D comprises the amino acid sequence of SEQ ID NO: 205; An antigen-binding molecule according to any one of claims 1 to 12 or an isolated antibody according to any one of claims 13 to 15.

17. 1. A pharmaceutical composition comprising: A therapeutically effective amount of an antigen-binding molecule according to any one of claims 1 to 12 or an isolated antibody according to any one of claims 13 to 15, and one or more pharma- ceutically acceptable vectors, diluents, buffers or excipients, Preferably, the composition further comprises at least one second therapeutic agent. Pharmaceutical compositions.

18. An isolated nucleic acid encoding the antigen-binding molecule of any one of claims 1 to 12 or the isolated antibody of any one of claims 13 to 15.

19. A host cell comprising the nucleic acid of claim 18.

20. A method for treating a disease, comprising administering to a subject an antigen-binding molecule according to any one of claims 1 to 12, an isolated antibody according to any one of claims 13 to 15, or a pharmaceutical composition according to claim 17, Preferably, the disease is a tumor, a cancer or an autoimmune disease; More preferably, the disease is multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, Waldenström's macroglobulinemia, plasma cell leukemia, light chain amyloidosis, precursor B-cell lymphoblastic leukemia, acute myeloid leukemia. , myelodysplastic syndrome, chronic lymphocytic leukemia, B-cell malignancies, chronic myeloid leukemia, hairy cell leukemia, blastic plasmacytoid dendritic cell neoplasm, Hodgkin's lymphoma, non-Hodgkin's lymphoma, marginal zone B-cell lymphoma, mucosa-associated lymphoid tissue lymphoma, plasma cell leukemia, anaplastic large cell lymphoma, ovarian cancer, lung cancer, gastric cancer, prostate cancer, renal cancer, liver cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, or melanoma, method.

Images