Pharmaceutical composition comprising bispecific antibody specifically binding to GPRC5d and CD3

Abstract

A pharmaceutical composition comprising a bispecific antibody specifically binding to GPRC5D and CD3. Specifically, the provided pharmaceutical composition comprises the bispecific antibody specifically binding to GPRC5D and CD3, and a buffer agent.

Description

A pharmaceutical composition comprising a bispecific antibody that specifically binds to GPRC5D and CD3 Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to GPRC5D and CD3 and its use. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

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

[0004] GPRC5D is a G protein-coupled orphan receptor, a seven-transmembrane protein that is specifically overexpressed in multiple myeloma patients and correlates with poor prognosis, whereas it is barely expressed in most normal tissues. Furthermore, GPR5CD expression is significantly higher in both newly diagnosed and relapsed / refractory MM patients compared to healthy controls. Furthermore, GPRC5D expression remains unchanged in various aberrant cell types, including those with 1(q)gain and 13(q)del. This association between GPRC5D overexpression and cancer suggests that GPRC5D may serve as an excellent therapeutic target for cancer.

[0005] There are currently many therapeutic drugs for MM in clinical practice, including immunomodulators (IMIDs), protease inhibitors (PIs), and monoclonal antibodies. These drugs have indeed improved the clinical condition of MM patients, but these therapies all have their own disadvantages. For example, IMIDs can cause birth defects, neurotoxicity, and severe thrombosis, while PIs are accompanied by a high risk of peripheral neurotoxicity. Monoclonal antibodies against CD38 and SLAMF7 can cause severe infections and anemia due to the reduction of red and white blood cells, and can also cause drug resistance due to antigen loss. Currently, there is no treatment that can completely cure MM, and patients will eventually relapse. Therefore, the development of a new targeted immunotherapy is of relatively important clinical significance.

[0006] Summary of the Invention

[0007] The present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to GPRC5D and CD3. The pharmaceutical composition has therapeutic activity and also has advantages such as good stability.

[0008] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to GPRC5D and CD3 and a buffer, wherein:

[0009] The bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen-binding module 1 that specifically binds to GPRC5D and an antigen-binding module 2 that specifically binds to CD3, wherein the antigen-binding module 1 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 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL;

[0010] in,

[0011] (i) the GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, 14, 41, 42 or 44, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16, or

[0012] The GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, 6 or 30, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 27, 8, 26, 28 or 29, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10; and

[0013] (ii) the CD3-VH comprises: a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 53; and the CD3-VL comprises: a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 56;

[0014] The buffer is histidine buffer, acetate buffer, citrate buffer, succinate buffer or phosphate buffer.

[0015] In some embodiments, the pharmaceutical composition as described above, wherein the buffer is histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer.

[0016] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the buffer is histidine-histidine hydrochloride buffer.

[0017] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the pH of the pharmaceutical composition is 5.0 to 7.0. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 6.0. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.5 to 6.0. In some embodiments, the pH of the pharmaceutical composition is about 5.5. In some embodiments, the pH of the pharmaceutical composition is about 5.6. In some embodiments, the pH of the pharmaceutical composition is about 5.7. When a point value is mentioned in this disclosure, it should be understood that the point value includes an error range. This error range is due to factors such as laboratory environment, human operation, instrumentation, methodology, measurement error, etc. Taking pH as an example, when the measured value is about 5.5, it should be understood that it includes an error range. As an example, when a preparation is measured using an industrial pH meter, "about 5.5" means 5.5±0.2 (i.e., a pH of 5.3 to 5.7).

[0018] In some embodiments, the pH of the pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or any range therebetween. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.6. In some embodiments, the pH of the pharmaceutical composition is 5.7.

[0019] Typically, the pH of the pharmaceutical composition obtained by replacing the buffer is almost identical to the buffer pH. Meanwhile, it is well known to those skilled in the art that in the process of pharmaceutical preparation, there may sometimes be a pH drift, but the drift of the pH of the pharmaceutical preparation is generally very small (within the range of ± 0.3). In some embodiments, the drift of the pH of the pharmaceutical preparation is within the range of ± 0.2. In some embodiments, the drift of the pH of the pharmaceutical preparation is within the range of ± 0.1. As an example, "a pH drift of 5.5" means that the drift of pH is within the range of 5.5 ± 0.3 (i.e., pH is 5.2 to 5.8), or the drift of pH is within the range of 5.5 ± 0.2 (i.e., pH is 5.3 to 5.7), or the drift of pH is within the range of 5.5 ± 0.1 (i.e., pH is 5.4 to 5.6).

[0020] In some embodiments, the pharmaceutical composition of any of the above items, wherein the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 1 mg / mL and 250 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 1 mg / mL and 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 1 mg / mL and 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 60 mg / mL and 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 80 mg / mL and 120 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 90 mg / mL and 110 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is about 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 60 mg / mL and 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 80 mg / mL and 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 100 mg / mL and 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is between 120 mg / mL and 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is about 1 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is about 2 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is about 5 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds GPRC5D and CD3 is about 60 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds GPRC5D and CD3 is about 150 mg / mL.In some embodiments, the concentration of the bispecific antibody that specifically binds GPRC5D and CD3 is 1 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 1 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 2 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 5 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 60 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds GPRC5D and CD3 is 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds GPRC5D and CD3 is 150 mg / mL.

[0021] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20, polysorbate 80), polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, Myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, etc. In some embodiments, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 80 or poloxamer 188. In some embodiments, the surfactant is polysorbate 80.

[0022] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the surfactant concentration is 0.01 mg / mL to 8.0 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the surfactant concentration is 0.01 mg / mL to 3.0 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the surfactant concentration is 0.01 mg / mL to 2.5 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the surfactant concentration is 0.01 mg / mL to 2.0 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the surfactant concentration is 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL to 0.6 mg / mL. In some embodiments, the surfactant concentration is 0.32 mg / mL to 0.48 mg / mL. In some embodiments, the surfactant concentration is 0.36mg / mL to 0.44mg / mL. In some embodiments, the surfactant concentration is about 0.2mg / mL. In some embodiments, the surfactant concentration is about 0.4mg / mL. In some embodiments, the surfactant concentration is about 0.6mg / mL. In some embodiments, the surfactant concentration is 0.01mg / mL, 0.05mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, 0.6mg / mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL or 1.0mg / mL, or any range between these point values. In some embodiments, the surfactant concentration is 0.4mg / mL.

[0023] In some embodiments, the surfactant is 0.01 mg / mL to 1.0 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.1 mg / mL to 0.8 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.2 mg / mL to 0.6 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.32 mg / mL to 0.48 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.36 mg / mL to 0.44 mg / mL of polysorbate 80. In some embodiments, the surfactant is about 0.4 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.2 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.4 mg / mL of polysorbate 80. In some embodiments, the surfactant is 0.6 mg / mL of polysorbate 80.

[0024] In some embodiments, the surfactant is 0.5 mg / mL to 8.0 mg / mL of PF68. In some embodiments, the surfactant is 0.5 mg / mL to 3.0 mg / mL of PF68. In some embodiments, the surfactant is 2.0 mg / mL of PF68.

[0025] In some embodiments, the pharmaceutical composition as described in any one of the above items comprises sugar. In some embodiments, the sugar is selected from the conventional composition (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. The sugar can be selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like.

[0026] In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose or trehalose. In some embodiments, the sugar is sucrose.

[0027] In some embodiments, the concentration of the sugar is between 10 mg / mL and 100 mg / mL. In some embodiments, the concentration of the sugar is between 30 mg / mL and 100 mg / mL. In some embodiments, the concentration of the sugar is between 64 mg / mL and 96 mg / mL. In some embodiments, the concentration of the sugar is between 68 mg / mL and 92 mg / mL. In some embodiments, the concentration of the sugar is between 72 mg / mL and 88 mg / mL. In some embodiments, the concentration of the sugar is about 30 mg / mL. In some embodiments, the concentration of the sugar is about 80 mg / mL. In some embodiments, the concentration of the sugar is about 100 mg / mL. In some embodiments, the concentration of the saccharide is, by way of non-limiting example, 10 mg / mL, 20 mg / mL, 30 mg / mL, 35 mg / mL, 37.5 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, and any ranges therebetween. In some embodiments, the concentration of the saccharide is 30 mg / mL.

[0028] In some embodiments, the concentration of the sugar is 80 mg / mL. In some embodiments, the concentration of the sugar is 100 mg / mL.

[0029] In some embodiments, the sugar is 10 mg / mL to 100 mg / mL of sucrose. In some embodiments, the sugar is 30 mg / mL to 100 mg / mL of sucrose. In some embodiments, the sugar is 64 mg / mL to 96 mg / mL of sucrose. In some embodiments, the sugar is 68 mg / mL to 92 mg / mL of sucrose. In some embodiments, the sugar is 72 mg / mL to 88 mg / mL of sucrose. In some embodiments, the sugar is about 30 mg / mL of sucrose. In some embodiments, the sugar is about 80 mg / mL of sucrose. In some embodiments, the sugar is about 100 mg / mL of sucrose.

[0030] In some embodiments, the concentration of the buffer is 5mM to 100mM. In some embodiments, the concentration of the buffer is 5mM to 50mM. In some embodiments, the concentration of the buffer is 10mM to 50mM. In some embodiments, the concentration of the buffer is 5mM to 30mM. In some embodiments, the concentration of the buffer is 5mM to 25mM. In some embodiments, the concentration of the buffer is 5mM to 20mM. In some embodiments, the concentration of the buffer is 8mM to 20mM. In some embodiments, the concentration of the buffer is 14mM to 20mM. In some embodiments, the concentration of the buffer is about 5mM. In some embodiments, the concentration of the buffer is about 14mM. In some embodiments, the concentration of the buffer is about 20mM. In some embodiments, the concentration of the buffer is about 50mM. In some embodiments, the concentration of the buffer is 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, and any range between these point values. In some embodiments, the concentration of the buffer is 5mM. In some embodiments, the concentration of the buffer is 14mM. In some embodiments, the concentration of the buffer is 20mM. In some embodiments, the concentration of the buffer is 50mM.

[0031] In some embodiments, the buffer is 5mM to 100mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 5mM to 50mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 5mM to 30mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 5mM to 25mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 5mM to 20mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 8mM to 20mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is about 5mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is about 14mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is about 20mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is about 50 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 5 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 14 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 20 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 50 mM histidine-histidine hydrochloride buffer.

[0032] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the pharmaceutical composition further comprises an excipient. In some embodiments, the excipient is ethylenediaminetetraacetic acid disodium salt or its hydrate, arginine hydrochloride, glycine, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, or calcium chloride. In some embodiments, the excipient is ethylenediaminetetraacetic acid disodium salt dihydrate (also known as "disodium edetate" or "EDTA-2Na").

[0033] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the excipient is 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL to 0.2 mg / mL. In some embodiments, the concentration of the excipient is 0.08 mg / mL to 0.12 mg / mL. In some embodiments, the concentration of the excipient is 0.09 mg / mL to 0.11 mg / mL. In some embodiments, the concentration of the excipient is about 0.01 mg / mL. In some embodiments, the concentration of the excipient is about 0.1 mg / mL. In some embodiments, the concentration of the excipient is about 1 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL, 0.03 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, and any range between these point values. In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the concentration of the excipient is 0.01 mg / mL. In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the concentration of the excipient is 0.1 mg / mL. In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the concentration of the excipient is 1 mg / mL.

[0034] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the excipient is 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is 0.01 mg / mL to 0.2 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is 0.08 mg / mL to 0.12 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is 0.09 mg / mL to 0.11 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is about 0.01 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is about 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is 0.01 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate. In some embodiments, the excipient is 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate.

[0035] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3,

[0036] The GPRC5D-VH comprises: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, and the GPRC5D-VL comprises: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16,

[0037] and the CD3-VH having: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 53; and the CD3-VL having: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0038] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3,

[0039] The GPRC5D-VH comprises: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and the GPRC5D-VL comprises: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 27, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10;

[0040] and the CD3-VH having: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 53; and the CD3-VL having: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0041] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3,

[0042] The GPRC5D-VH has: GPRC5D-HCDR1 as shown in SEQ ID NO: 11, GPRC5D-HCDR2 as shown in SEQ ID NO: 12, GPRC5D-HCDR3 as shown in SEQ ID NO: 13, and the GPRC5D-VL has: GPRC5D-LCDR1 as shown in SEQ ID NO: 43, GPRC5D-LCDR2 as shown in SEQ ID NO: 15, and GPRC5D-LCDR3 as shown in SEQ ID NO: 16,

[0043] And the CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 51, CD3-HCDR2 as shown in SEQ ID NO: 52, CD3-HCDR3 as shown in SEQ ID NO: 53, and the CD3-VL has: CD3-LCDR1 as shown in SEQ ID NO: 54, CD3-LCDR2 as shown in SEQ ID NO: 55, and CD3-LCDR3 as shown in SEQ ID NO: 56.

[0044] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3,

[0045] The GPRC5D-VH has: GPRC5D-HCDR1 as shown in SEQ ID NO: 5, GPRC5D-HCDR2 as shown in SEQ ID NO: 31, GPRC5D-HCDR3 as shown in SEQ ID NO: 7, and the GPRC5D-VL has: GPRC5D-LCDR1 as shown in SEQ ID NO: 27, GPRC5D-LCDR2 as shown in SEQ ID NO: 9, and GPRC5D-LCDR3 as shown in SEQ ID NO: 10;

[0046] And the CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 51, CD3-HCDR2 as shown in SEQ ID NO: 52, CD3-HCDR3 as shown in SEQ ID NO: 53, and the CD3-VL has: CD3-LCDR1 as shown in SEQ ID NO: 54, CD3-LCDR2 as shown in SEQ ID NO: 55, and CD3-LCDR3 as shown in SEQ ID NO: 56.

[0047] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3,

[0048] The GPRC5D-HCDR1, GPRC5D-HCDR2, GPRC5D-HCDR3, GPRC5D-LCDR1, GPRC5D-LCDR2 and GPRC5D-LCDR3, and

[0049] The CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering rules; preferably, they are defined according to the Kabat numbering rules.

[0050] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody specifically binding to GPRC5D and CD3, wherein:

[0051] (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, 32 or 33, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 34, 32 or 33, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39, 35, 36, 37, 38, 40, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 39, 35, 36, 37, 38, 40; or

[0052] the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4;

[0053] and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 58; or

[0054] (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 19, 17 or 18, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 19, 17 or 18, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 23, 20, 21, 22, 24 or 25, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 23, 20, 21, 22, 24 or 25; or

[0055] the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2;

[0056] and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 57 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 58.

[0057] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody specifically binding to GPRC5D and CD3, wherein:

[0058] (i) 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; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 58; or

[0059] (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 19, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 23; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 58.

[0060] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3, wherein:

[0061] (i) the amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 34, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 39; and the amino acid sequence of the CD3-VH is shown in SEQ ID NO: 57, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 58; or

[0062] (ii) the amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 19, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 23; and the amino acid sequence of the CD3-VH is shown in SEQ ID NO: 57, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 58.

[0063] In some embodiments, the pharmaceutical composition according to any one of the above items, wherein the antigen binding moiety that specifically binds to GPRC5D is Fab; the antigen binding moiety that specifically binds to CD3 is a replaced Fab; the replaced Fab is a Fab in which the original CH1 and CL are replaced by the Obscurin chain and the Titin chain, respectively;

[0064] Preferably, the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 83, and the Titin chain comprises the amino acid sequence of SEQ ID NO: 84.

[0065] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3 has a structure of Formula 2-0G4S or Formula 4-0G4S; wherein, the structural schematic diagram of Formula 2-0G4S is shown in Figure 1, and the structural schematic diagram of Formula 4-0G4S is shown in Figure 2.

[0066] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen binding moiety 1 that specifically binds to GPRC5D and an antigen binding moiety 2 that specifically binds to CD3, the antigen binding moiety 1 that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, the antigen binding moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in Figure 2;

[0067] in,

[0068] The GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, 14, 41, 42 or 44, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16;

[0069] and the CD3-VH having: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 53; and the CD3-VL having: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0070] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen binding moiety 1 that specifically binds to GPRC5D and an antigen binding moiety 2 that specifically binds to CD3, the antigen binding moiety 1 that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, the antigen binding moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in Figure 1;

[0071] wherein: the GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, 6 or 30, a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 27, 8, 26, 28 or 29, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10; and

[0072] The CD3-VH comprises: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 53; and the CD3-VL comprises: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.

[0073] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0074] The bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen-binding moiety 1 that specifically binds to GPRC5D and an antigen-binding moiety 2 that specifically binds to CD3, wherein the antigen-binding moiety 1 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 moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in FIG2 ;

[0075] in,

[0076] The GPRC5D-VH has: GPRC5D-HCDR1 as shown in SEQ ID NO: 11, GPRC5D-HCDR2 as shown in SEQ ID NO: 12, GPRC5D-HCDR3 as shown in SEQ ID NO: 13, and the GPRC5D-VL has: GPRC5D-LCDR1 as shown in SEQ ID NO: 43, GPRC5D-LCDR2 as shown in SEQ ID NO: 15, and GPRC5D-LCDR3 as shown in SEQ ID NO: 16,

[0077] And the CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 51, CD3-HCDR2 as shown in SEQ ID NO: 52, CD3-HCDR3 as shown in SEQ ID NO: 53, and the CD3-VL has: CD3-LCDR1 as shown in SEQ ID NO: 54, CD3-LCDR2 as shown in SEQ ID NO: 55, and CD3-LCDR3 as shown in SEQ ID NO: 56.

[0078] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0079] The bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen-binding moiety 1 that specifically binds to GPRC5D and an antigen-binding moiety 2 that specifically binds to CD3, wherein the antigen-binding moiety 1 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 moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in FIG1 ;

[0080] The GPRC5D-VH has: GPRC5D-HCDR1 as shown in SEQ ID NO: 5, GPRC5D-HCDR2 as shown in SEQ ID NO: 31, GPRC5D-HCDR3 as shown in SEQ ID NO: 7, and the GPRC5D-VL has: GPRC5D-LCDR1 as shown in SEQ ID NO: 27, GPRC5D-LCDR2 as shown in SEQ ID NO: 9, and GPRC5D-LCDR3 as shown in SEQ ID NO: 10;

[0081] And the CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 51, CD3-HCDR2 as shown in SEQ ID NO: 52, CD3-HCDR3 as shown in SEQ ID NO: 53, and the CD3-VL has: CD3-LCDR1 as shown in SEQ ID NO: 54, CD3-LCDR2 as shown in SEQ ID NO: 55, and CD3-LCDR3 as shown in SEQ ID NO: 56.

[0082] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0083] The bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen-binding moiety 1 that specifically binds to GPRC5D and an antigen-binding moiety 2 that specifically binds to CD3, wherein the antigen-binding moiety 1 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 moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in FIG2 .

[0084] wherein the amino acid sequence of GPRC5D-VH is shown in SEQ ID NO: 34, and the amino acid sequence of said GPRC5D-VL is shown in SEQ ID NO: 39; and

[0085] The amino acid sequence of the CD3-VH is shown in SEQ ID NO: 57, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 58.

[0086] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein: the bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen-binding moiety 1 that specifically binds to GPRC5D and an antigen-binding moiety 2 that specifically binds to CD3, the antigen-binding moiety 1 that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, the antigen-binding moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in Figure 1,

[0087] The amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 19, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 23; and the amino acid sequence of the CD3-VH is shown in SEQ ID NO: 57, and the amino acid sequence of CD3-VL is shown in SEQ ID NO: 58.

[0088] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0089] The bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen-binding moiety 1 that specifically binds to GPRC5D and an antigen-binding moiety 2 that specifically binds to CD3, wherein the antigen-binding moiety 1 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 moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in FIG2 .

[0090] wherein the amino acid sequence of GPRC5D-VH is shown in SEQ ID NO: 34, and the amino acid sequence of said GPRC5D-VL is shown in SEQ ID NO: 39; and

[0091] The amino acid sequence of the CD3-VH is shown in SEQ ID NO: 57, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 58,

[0092] The Obscurin chain comprises the amino acid sequence of SEQ ID NO: 83, and the Titin chain comprises the amino acid sequence of SEQ ID NO: 84.

[0093] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0094] The bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen-binding moiety 1 that specifically binds to GPRC5D and an antigen-binding moiety 2 that specifically binds to CD3, wherein the antigen-binding moiety 1 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 moiety 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and has the structure shown in FIG1 .

[0095] The amino acid sequence of the GPRC5D-VH is shown in SEQ ID NO: 19, and the amino acid sequence of the GPRC5D-VL is shown in SEQ ID NO: 23; and the amino acid sequence of the CD3-VH is shown in SEQ ID NO: 57, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 58,

[0096] The Obscurin chain comprises the amino acid sequence of SEQ ID NO: 83, and the Titin chain comprises the amino acid sequence of SEQ ID NO: 84.

[0097] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0098] The bispecific antibody that specifically binds to GPRC5D and CD3, which has a first chain as shown in SEQ ID NO: 61, a second chain as shown in SEQ ID NO: 60, a third chain as shown in SEQ ID NO: 67, and a fourth chain as shown in SEQ ID NO: 68; or

[0099] It has a first strand as shown in SEQ ID NO:65, a second strand as shown in SEQ ID NO:66, a third strand as shown in SEQ ID NO:59, and a fourth strand as shown in SEQ ID NO:60.

[0100] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0101] The bispecific antibody that specifically binds to GPRC5D and CD3 has a first chain as shown in SEQ ID NO: 61, a second chain as shown in SEQ ID NO: 60, a third chain as shown in SEQ ID NO: 67, and a fourth chain as shown in SEQ ID NO: 68.

[0102] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0103] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0104] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0105] (c) 10 mg / mL to 100 mg / mL of sugar,

[0106] (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt or a hydrate thereof, and

[0107] (e) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0108] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0109] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0110] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0111] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0112] (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0113] (e) 5 mM to 50 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0114] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0115] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0116] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80,

[0117] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0118] (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0119] (e) 5 mM to 50 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0120] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0121] (a) 60 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0122] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0123] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0124] (d) 0.01 mg / mL to 0.2 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0125] (e) 5 mM to 50 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0126] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0127] (a) 60 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0128] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80,

[0129] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0130] (d) 0.01 mg / mL to 0.2 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0131] (e) 5 mM to 30 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0132] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0133] (a) 80 mg / mL to 120 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0134] (b) 0.32 mg / mL to 0.48 mg / mL of polysorbate 80,

[0135] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0136] (d) 0.08 mg / mL to 0.12 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0137] (e) 8 mM to 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5 to 6.0.

[0138] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0139] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0140] (b) 0.36 mg / mL to 0.44 mg / mL of polysorbate 80,

[0141] (c) 72 mg / mL to 88 mg / mL of sucrose,

[0142] (d) 0.09 mg / mL to 0.11 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0143] (e) 8 mM to 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5 to 6.0.

[0144] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0145] (a) about 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0146] (b) about 0.4 mg / mL of polysorbate 80,

[0147] (c) about 80 mg / mL of sucrose,

[0148] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0149] (e) about 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.6.

[0150] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0151] (a) about 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0152] (b) about 0.4 mg / mL of polysorbate 80,

[0153] (c) about 80 mg / mL of sucrose,

[0154] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0155] (e) about 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.6.

[0156] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0157] (a) about 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0158] (b) about 0.4 mg / mL of polysorbate 80,

[0159] (c) about 80 mg / mL of sucrose,

[0160] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0161] (e) about 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.6.

[0162] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0163] (a) 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0164] (b) 0.4 mg / mL of polysorbate 80,

[0165] (c) 80 mg / mL sucrose,

[0166] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0167] (e) 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.6.

[0168] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0169] (a) 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0170] (b) 0.4 mg / mL of polysorbate 80,

[0171] (c) 80 mg / mL sucrose,

[0172] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0173] (e) 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.6.

[0174] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0175] (a) 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0176] (b) 0.4 mg / mL of polysorbate 80,

[0177] (c) 80 mg / mL sucrose,

[0178] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0179] (e) 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.6.

[0180] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0181] (a) about 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0182] (b) about 0.4 mg / mL of polysorbate 80,

[0183] (c) about 80 mg / mL of sucrose,

[0184] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0185] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0186] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0187] (a) about 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0188] (b) about 0.4 mg / mL of polysorbate 80,

[0189] (c) about 80 mg / mL of sucrose,

[0190] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0191] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0192] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0193] (a) about 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0194] (b) about 0.4 mg / mL of polysorbate 80,

[0195] (c) about 80 mg / mL of sucrose,

[0196] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0197] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0198] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0199] (a) 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0200] (b) 0.4 mg / mL of polysorbate 80,

[0201] (c) 80 mg / mL sucrose,

[0202] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0203] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0204] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0205] (a) 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0206] (b) 0.4 mg / mL of polysorbate 80,

[0207] (c) 80 mg / mL sucrose,

[0208] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0209] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0210] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0211] (a) 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0212] (b) 0.4 mg / mL of polysorbate 80,

[0213] (c) 80 mg / mL sucrose,

[0214] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0215] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0216] In some embodiments, the pharmaceutical composition as described above is a liquid formulation. In some embodiments, the solvent in the liquid formulation is water.

[0217] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the pharmaceutical compositions described above after reconstitution.

[0218] The present disclosure also provides a lyophilized preparation, which is a lyophilized preparation of the pharmaceutical composition as described in any one of the above items.

[0219] The present disclosure also provides a method for preparing a lyophilized formulation, comprising freeze-drying the pharmaceutical composition as described in any one of the above items. In some embodiments, the freeze-drying as described in any one of the above items sequentially comprises the steps of pre-freezing, primary drying, and secondary drying.

[0220] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0221] The present disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted the lyophilized preparation as described in any one of the above items.

[0222] The present disclosure also provides a reconstituted solution, which is a reconstituted preparation of any of the above lyophilized preparations.

[0223] In some embodiments, the components and contents of the reconstituted solution as described in any of the above items are the same as those of the aforementioned pharmaceutical composition.

[0224] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0225] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0226] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0227] (c) 10 mg / mL to 100 mg / mL of sugar,

[0228] (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt or a hydrate thereof, and

[0229] (e) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0230] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0231] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0232] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0233] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0234] (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0235] (e) 5 mM to 50 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0236] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0237] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0238] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80,

[0239] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0240] (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0241] (e) 5 mM to 50 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0242] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0243] (a) 60 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0244] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0245] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0246] (d) 0.01 mg / mL to 0.2 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0247] (e) 5 mM to 50 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0248] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0249] (a) 60 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3,

[0250] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80,

[0251] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0252] (d) 0.01 mg / mL to 0.2 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0253] (e) 5 mM to 30 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0254] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0255] (a) 80 mg / mL to 120 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0256] (b) 0.32 mg / mL to 0.48 mg / mL of polysorbate 80,

[0257] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0258] (d) 0.08 mg / mL to 0.12 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0259] (e) 8 mM to 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5 to 6.0.

[0260] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0261] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0262] (b) 0.36 mg / mL to 0.44 mg / mL of polysorbate 80,

[0263] (c) 72 mg / mL to 88 mg / mL of sucrose,

[0264] (d) 0.09 mg / mL to 0.11 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0265] (e) 8 mM to 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5 to 6.0.

[0266] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0267] (a) about 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0268] (b) about 0.4 mg / mL of polysorbate 80,

[0269] (c) about 80 mg / mL of sucrose,

[0270] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0271] (e) about 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.6.

[0272] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0273] (a) about 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0274] (b) about 0.4 mg / mL of polysorbate 80,

[0275] (c) about 80 mg / mL of sucrose,

[0276] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0277] (e) about 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.6.

[0278] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0279] (a) about 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0280] (b) about 0.4 mg / mL of polysorbate 80,

[0281] (c) about 80 mg / mL of sucrose,

[0282] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0283] (e) about 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.6.

[0284] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0285] (a) 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0286] (b) 0.4 mg / mL of polysorbate 80,

[0287] (c) 80 mg / mL sucrose,

[0288] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0289] (e) 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.6.

[0290] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0291] (a) 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0292] (b) 0.4 mg / mL of polysorbate 80,

[0293] (c) 80 mg / mL sucrose,

[0294] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0295] (e) 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.6.

[0296] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0297] (a) 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0298] (b) 0.4 mg / mL of polysorbate 80,

[0299] (c) 80 mg / mL sucrose,

[0300] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0301] (e) 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.6.

[0302] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0303] (a) about 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0304] (b) about 0.4 mg / mL of polysorbate 80,

[0305] (c) about 80 mg / mL of sucrose,

[0306] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0307] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0308] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0309] (a) about 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0310] (b) about 0.4 mg / mL of polysorbate 80,

[0311] (c) about 80 mg / mL of sucrose,

[0312] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0313] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0314] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0315] (a) about 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0316] (b) about 0.4 mg / mL of polysorbate 80,

[0317] (c) about 80 mg / mL of sucrose,

[0318] (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0319] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0320] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0321] (a) 2 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0322] (b) 0.4 mg / mL of polysorbate 80,

[0323] (c) 80 mg / mL sucrose,

[0324] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0325] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0326] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0327] (a) 60 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0328] (b) 0.4 mg / mL of polysorbate 80,

[0329] (c) 80 mg / mL sucrose,

[0330] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0331] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0332] In some embodiments, the reconstituted solution as described in any of the above items comprises the following components:

[0333] (a) 100 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3,

[0334] (b) 0.4 mg / mL of polysorbate 80,

[0335] (c) 80 mg / mL sucrose,

[0336] (d) 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and

[0337] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0338] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation, or an intramuscular injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is an intravenous injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is a subcutaneous injection preparation.

[0339] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for intravenous injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for subcutaneous injection.

[0340] In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation as described in any of the above items is used to prepare a drug for intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection. In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation as described in any of the above items is used to prepare a drug for intravenous injection. In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation as described in any of the above items is used to prepare a drug for subcutaneous injection.

[0341] The present disclosure also provides a drug kit comprising at least one container, each container independently containing the pharmaceutical composition as described in any one of the above items, the lyophilized formulation as described in any one of the above items, or the reconstituted solution as described in any one of the above items.

[0342] In some embodiments, the present disclosure also provides a method for diagnosing, treating, or alleviating a condition in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition, lyophilized formulation, reconstituted solution, or kit as described above.

[0343] In some embodiments, the present disclosure also provides a method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition as described in any one of the above, the lyophilized formulation as described in any one of the above, the reconstituted solution as described in any one of the above, or the drug kit as described in any one of the above.

[0344] In some embodiments, the present disclosure further provides a pharmaceutical composition as described in any of the above, a lyophilized formulation as described in any of the above, a reconstituted solution as described in any of the above, or a drug kit as described in any of the above, for use in treating or preventing a disease.

[0345] In one aspect, the present disclosure also provides use of the pharmaceutical composition as described in any of the preceding items, the lyophilized preparation as described in any of the preceding items, the reconstituted solution as described in any of the preceding items, or the drug kit as described in any of the preceding items in the preparation of a drug for preventing or treating a disease.

[0346] In some embodiments, the disease as described in any of the preceding items is selected from proliferative diseases (including tumors, cancer) and autoimmune diseases.

[0347] In some embodiments, the disease as described in any of the preceding items is a hematological malignancy or a solid tumor. In some embodiments, the hematological malignancy is selected from multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy (MGUS) (e.g., monoclonal gammopathy of undetermined 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 (ALA), leukemia, and the like. In some embodiments, the solid tumor is selected from ovarian cancer, lung cancer (non-small cell lung cancer and small cell lung cancer), gastric cancer, prostate cancer, kidney cancer, liver cancer, colorectal cancer (such as colon cancer and rectal cancer), esophageal cancer (esophageal cancer), bladder cancer, cervical cancer and melanoma.

[0348] In some embodiments, the disease is selected from multiple myeloma, relapsed / refractory multiple myeloma, smoldering multiple myeloma, monoclonal gammopathy (MGUS) (e.g., monoclonal gammopathy of undetermined significance), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, plasma cell leukemia, light chain amyloidosis (AL), precursor B-cell lymphoblastic 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 lymphoma, non-Hodgkin 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, kidney cancer, liver cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, and melanoma.

[0349] In some embodiments, the disease is multiple myeloma.

[0350] In some embodiments, the disease is relapsed / refractory multiple myeloma.

[0351] In some embodiments, the aforementioned disease is a disease associated with GPRC5D; in some embodiments, the aforementioned disease is a disease expressing GPRC5D. BRIEF DESCRIPTION OF THE DRAWINGS

[0352] Figure 1 is a schematic structural diagram of formula 2-0G4S;

[0353] FIG2 is a schematic diagram of the structure of formula 4-0G4S. DETAILED DESCRIPTION

[0354] the term

[0355] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0356] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0357] The term "antibody" is used in the broadest sense and covers various 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), as long as they exhibit the desired antigen-binding activity. "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric proteins of approximately 150,000 daltons, composed of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0358] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures. According to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. According to the number of antigen-binding regions, it can be divided into bispecific antibodies of divalent, trivalent, tetravalent or more valence. According to whether the structure is symmetrical, it can be divided into bispecific antibodies of symmetrical structure and bispecific antibodies of asymmetrical structure. Among them, fragment-type bispecific antibodies, such as Fab fragments lacking Fc fragments, form bispecific antibodies by combining two or more Fab fragments into one molecule. It has low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab′)2, scFv-Fab, (scFv)2-Fab, etc. IgG-like bispecific antibodies (e.g., with an Fc fragment) have a relatively large molecular weight. The Fc fragment helps purify the antibody and improve its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 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 and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11;2019:4516041).

[0359] The term "variable region" or "variable domain" refers to the domain of an antigen-binding molecule that binds to an antigen. Herein, the heavy chain variable region in the antigen-binding moiety 1 that specifically binds to HGFR is labeled M-VH, and the light chain variable region is labeled M-VL; the heavy chain variable region in the antigen-binding moiety 2 that specifically binds to EGFR is labeled E-VH, and the light chain variable region is labeled E-VL. VH and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Herein, the three CDR regions in M-VH are designated M-HCDR1, M-HCDR2, and M-HCDR3; the three CDR regions in M-VL are designated M-LCDR1, M-LCDR2, and M-LCDR3; the three CDR regions in E-VH are designated E-HCDR1, E-HCDR2, and E-HCDR3; and the three CDR regions in E-VL are designated E-LCDR1, E-LCDR2, and E-LCDR3. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0360] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "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), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. The correspondence between various numbering systems is well known to those skilled in the art. The numbering convention of the present disclosure is shown in Table 1 below.

[0361] Table 1. Relationships between CDR numbering systems

[0362] Unless otherwise indicated, the variable region and CDR sequences in the disclosed embodiments are all subject to the "Kabat" numbering convention. Although the Kabat numbering convention is used to define amino acid residues in specific embodiments, corresponding technical solutions provided by other numbering systems will be considered equivalent technical solutions.

[0363] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the antigen-binding ability of the intact 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 formed from antibody fragments.

[0364] Fab refers to a protein composed of VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain) of an immunoglobulin.

[0365] Fv refers to the antigen-binding domain composed of the VH and VL of an immunoglobulin.

[0366] In the present disclosure, in some embodiments, the first Fab has a structure of Fab. In the replaced Fab, CH1 and CL are replaced by titin chain or obscurin chain, respectively.

[0367] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise indicated, the numbering convention for the Fc region is the EU index.

[0368] The term "Titin chain" refers to a 78-118 amino acid fragment of the titin protein, which comprises a peptide segment of the titin Ig-like 152 domain or a functional variant thereof. The titin chain is capable of forming a dimerization complex with the obscurin chain.

[0369] The term "obscurin chain" refers to an 87-117 amino acid fragment of the obscurin protein that contains a peptide fragment of the obscurin Ig-like 1 domain or a functional variant thereof; or refers to a 78-118 amino acid fragment of the obscurin-like 1 protein that contains a peptide fragment of the obscurin-like Ig-like 1 domain or a functional variant thereof. The obscurin chain is capable of binding to the titin chain to form a dimerization complex. The titin chain and obscurin chain disclosed herein can be used to replace the CH1 and CL in Fab to form a replaced Fab (Fab-S). This replacement does not affect the binding of the antigen-binding molecule to the antigen.

[0370] The terms "capable of specifically binding", "specific binding" or "binding" refer to the ability of an antibody to bind to an antigen or epitope with a higher affinity than to other antigens or epitopes. - 7 M or less (e.g., about 1×10 -8 In some embodiments, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or However, an antibody that specifically binds to an antigen or an epitope thereof may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, for example, Macaca fasciculata (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp), or marmoset (Callithrix jacchus) (common marmoset, marmoset).

[0371] The term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to a target antigen. Antigen binding moieties include antibodies and fragments thereof as described herein. Specific antigen binding moieties include the antigen binding domain of an antibody, which comprises an antibody heavy chain variable region and an antibody light chain variable region. The term "antigen binding moiety that specifically binds to HGFR" refers to a module that can bind to HGFR or its epitope with sufficient affinity. In certain embodiments, the antigen binding moiety that specifically binds to HGFR has the following equilibrium dissociation constant (KD): <about 1 μM, <about 100 nM or <about 10 nM or less, which is measured by the Biacore method. In certain embodiments, the antigen binding moiety that specifically binds to HGFR binds to a conserved epitope in HGFR from different species. The term "antigen binding moiety that specifically binds to EGFR" refers to a module that can bind to EGFR or its epitope with sufficient affinity so that the molecule containing the module can be used as a diagnostic and / or therapeutic agent targeting EGFR. In certain embodiments, the antigen binding moiety that specifically binds to EGFR has an equilibrium dissociation constant (KD) of < about 1 μM, < about 100 nM, < about 10 nM or less as measured by Biacore. In certain embodiments, the antigen binding moiety that specifically binds to EGFR binds to a conserved epitope in EGFR from different species. The antigen binding moiety comprises an antibody fragment as defined herein, such as a Fab, a replaced Fab, or a Fv.

[0372] In this context, the ordinal numbers in "antigen binding module 1", "antigen binding module 2", "linker 1" and "linker 2" are only used to distinguish different technical features and chemical entities; they do not limit any order, level or quantity.

[0373] The term "linker" refers to a connecting unit that connects two polypeptide fragments. In this article, the linkers appearing in the same or different structural formulas can be the same or different. The linker can be a peptide linker that contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used in this article can be the same or different. When "-" appears in the structural formula, it means that the units on both sides are directly connected by a covalent bond. When the term "bond" appears in a structural unit, it means that the units on both sides of the unit are directly connected.

[0374] Unless otherwise specified, the variable region and CDR sequences in the disclosed examples are all numbered using the "Kabat" convention.

[0375] A "pharmaceutical composition" refers to a composition containing one or more bispecific antibodies described herein, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and enabling its biological activity. As used herein, the terms "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0376] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0377] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods.

[0378] It will be understood by those skilled in the art that when reference is made to a numerical range, a cutoff value, or a specific value, "about" can be expressed as being within one or more than one standard deviation. Alternatively, "about" can be expressed as being within a range of up to 20% (i.e., ± 20%). Since many numerical values ​​used herein are determined experimentally, it will be understood by those skilled in the art that such determinations can differ between different experiments and typically differ between different experiments. Due to this inherent difference, it is believed that the values ​​used herein should not be overly limited. Therefore, the term "about" is used to encompass variations of ± 20% or less, ± 10% or less, ± 5% or less, ± 1% or less, ± 0.5% or less, or ± 0.1% or less from a specified value.

[0379] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0380] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, histidine-histidine sulfate, and the like, with histidine-histidine hydrochloride buffer being preferred. Histidine-histidine hydrochloride buffer can be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0381] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0382] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0383] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include citric acid-disodium hydrogen phosphate, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is citric acid-disodium hydrogen phosphate.

[0384] An "acetate buffer" is a buffer comprising acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

[0385] "Poloxamer" is a block copolymer of ethylene oxide and propylene oxide that is water-soluble and used as a surfactant in pharmaceutical formulations. Examples of poloxamers include poloxamer 188.

[0386] "Edetate disodium" (CAS No. 6381-92-6, the edetate disodium described in this disclosure is purchased from SIGMA.) The company's official website lists its aliases as ethylenediaminetetraacetic acid disodium salt dihydrate, EDTA disodium salt, EDTA-Na2, ethylenediaminetetraacetic acid Na2, edetate disodium salt dihydrate, disodium edetate dihydrate, and edetate calcium sodium. Therefore, "edetate disodium," "edetate disodium salt dihydrate," "EDTA-2Na," and "edetate disodium" are used interchangeably in this disclosure, and their substance is essentially the substance with CAS No. 6381-92-6.

[0387] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0388] The pharmaceutical compositions disclosed herein can achieve a stable effect: the antibody therein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0389] A stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 1 month, at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for a period of time, including 1 month, 3 months, or 6 months. They also include those that exhibit the desired characteristics after storage at 40°C for a period of time, including 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include: typically no more than about 10%, preferably no more than about 5%, of the antibody aggregates or degrades as measured by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly opalescent. The concentration, pH, weight, and molecular osmotic pressure of the formulation vary by no more than ±10%, preferably no more than ±5%. The formulation typically forms no more than about 10%, preferably no more than about 5%, of aggregates.

[0390] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0391] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0392] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0393] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions applied to the treated individual and can be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and resolving or improving the prognosis. In some embodiments, the antigen binding molecules of the present disclosure are used to delay the formation of the disease or slow the progression of the disease.

[0394] The details of one or more embodiments of the present disclosure are set forth in the above description. Although any methods and materials similar or identical to those described herein can be used to implement or test the present disclosure, preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will be apparent from the description and claims. In the description and claims, unless the context clearly indicates otherwise, the singular includes the case of plural referents. Unless otherwise defined, all technical and scientific terms used herein have the common meaning understood by those of ordinary skill in the art to which the present disclosure belongs. All patents and publications cited in the description are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the present disclosure. These examples should not be construed in any way to limit the scope of the present disclosure, which is defined by the claims.

[0395] Example - Preparation of a bispecific antibody that specifically binds to GPRC5D and CD3

[0396] PCT / CN2023 / 081237 (application date: 2023.03.14; priority patent application number: CN202210245308.7) is incorporated into this disclosure in its entirety by reference.

[0397] Example 1. Construction and identification of cell lines expressing recombinant human GPRC5D and cynomolgus monkey GPRC5D

[0398] To screen antibodies that can bind 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 gene of human GPRC5D or cynomolgus monkey GPRC5D was cloned into the mammalian cell expression vector pCDH, and HEK293T cells were co-transfected with pVSV-G, pCMV-dR8.91, and pCDH-humanGPRC5D or pCDH-cynoGPRC5D plasmids. CRL-11268) packaged virus, and 48 hours after transfection, the virus was collected and infected CHOK1 cells ( CCL-61) and HEK293 cells ( CRL-1573). Monoclonal cells highly expressing human GPRC5D or cynomolgus macaque GPRC5D were obtained by flow cytometry 72 hours after infection.

[0399] Example 2. Preparation of mouse anti-human GPRC5D monoclonal antibody

[0400] CHOK1-humanGPRC5D and CHOK1-cynoGPRC5D cells were used to cross-immunize SJL mice. After 7 immunizations, blood was collected to determine the titer of the antibody in the serum. Mice with high antibody titers in the serum and titers approaching a plateau were selected for spleen cell fusion. The fused hybridoma cells were plated in a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. The cell culture supernatant was detected by mirrorball and flow cytometry sorting technology (FACS). The screened positive clones were amplified, frozen, and subcloned two to three times until a single cell clone was obtained. The selected hybridoma clones were further prepared and purified using a serum-free cell culture method. The obtained hybridoma antibodies were detected by FACS for binding to CHOK1-humanGPRC5D, CHOK1-cynoGPRC5D, and CHOK1 cells (see Test Example 1 of this disclosure for the method), and hybridoma cell lines mAb1 and mAb24 with good human-monkey cross-binding activity and no non-specific binding were selected.

[0401] The sequences of the monoclonal antibodies from hybridoma cell lines mAb1 and mAb24 were cloned as follows: Hybridoma cells in logarithmic growth phase were harvested, RNA was extracted using Trizol (Invitrogen, Cat# 15596-018), and reverse transcribed into cDNA. PCR amplification of the cDNA template was performed and the resulting DNA sequence was sequenced by a sequencing company. The resulting DNA sequence corresponds to the following amino acid sequence of the antibody variable region:

[0402] Amino acid sequence of the heavy chain variable region of mAb1:

[0403] Amino acid sequence of the light chain variable region of mAb1:

[0404] Amino acid sequence of the heavy chain variable region of mAb24:

[0405] Amino acid sequence of the light chain variable region of mAb24: Note: The underlined part is CDR according to Kabat numbering convention, and the rest are FR.

[0406] Table 2. CDRs of GPRC5D Antibody Note: The CDRs in the table are confirmed according to the Kabat numbering system.

[0407] Example 3. Humanized design of anti-human GPRC5D monoclonal antibody

[0408] Humanization of murine monoclonal antibodies is performed using methods well-documented in the field. Briefly, based on the representative VH / VL CDR structures of the murine antibody, homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) are searched from human germline databases. The murine antibody CDR regions are then transplanted onto a human template, and some VL and VH residues are mutated. The murine antibody constant region is then replaced with a human constant region to yield the final humanized molecule.

[0409] 1. Humanization of mAb1

[0410] The humanized heavy chain variable region of mAb1 uses IGHV3-21*01 for FR1, FR2, and FR3, and IGHJ6*01 for FR4. The light chain variable region uses IGKV2-29*02 for FR1, FR2, and FR3, and IGKJ2*01 for FR4. Optionally, amino acids at positions 49, 56, 57, and / or 93 of the heavy chain variable region of the humanized antibody are substituted, and / or amino acids at positions 2, 28, 29, and / or 45 of the light chain variable region of the humanized antibody are substituted.

[0411] Table 3. Amino acid substitutions of the humanized antibody mAb1 Note: Exemplarily, Q45K means that according to the Kabat numbering system, the Q at position 45 is mutated back to K. And so on.

[0412] The sequence of the antibody variable region is as follows (the underlined part is CDR, the same below):

[0413] >hu1H1 (SEQ ID NO: 17):

[0414] >hu1H2 (SEQ ID NO: 18):

[0415] >hu1H3 (SEQ ID NO: 19):

[0416] >hu1L1 (SEQ ID NO: 20):

[0417] >hu1L2 (SEQ ID NO: 21):

[0418] >hu1L3 (SEQ ID NO: 22):

[0419] >hu1L4 (SEQ ID NO: 23):

[0420] >hu1L5 (SEQ ID NO: 24):

[0421] >hu1L6 (SEQ ID NO: 25):

[0422] The amino acid sequences of the replaced CDRs in the humanized antibodies are shown in the table below:

[0423] Table 4. Amino acid sequences of hu1 replaced CDRs

[0424] 2. Humanization of mAb24

[0425] The humanized heavy chain variable region of mAb24 utilizes IGHV1-46*01 for FR1, FR2, and FR3, and IGHJ6*01 for FR4. The light chain variable region utilizes IGKV2-30*02 for FR1, FR2, and FR3, and IGKJ4*01 for FR4. Optionally, amino acid substitutions are made at positions 1, 24, 37, 69, 71, 73, and / or 93 of the heavy chain variable region of the humanized antibody; and / or amino acid substitutions are made at positions 4, 28, 29, 36, and / or 37 of the light chain variable region of the humanized antibody.

[0426] Table 5. Amino acid substitutions of humanized mAb 24 Note: Exemplarily, M4L means that according to the Kabat numbering system, position 4 M is mutated back to L. And so on.

[0427] The sequence of the antibody variable region is as follows:

[0428] >hu24H1 (SEQ ID NO: 32):

[0429] >hu24H2 (SEQ ID NO: 33):

[0430] >hu24H3 (SEQ ID NO: 34):

[0431] >hu24L1 (SEQ ID NO: 35):

[0432] >hu24L2 (SEQ ID NO: 36):

[0433] >hu24L3 (SEQ ID NO: 37):

[0434] >hu24L4 (SEQ ID NO: 38):

[0435] >hu24L5 (SEQ ID NO: 39):

[0436] >hu24L6 (SEQ ID NO: 40):

[0437] The amino acid sequences of the replaced CDRs in the humanized antibodies are shown in the table below:

[0438] Table 6. Amino acid sequences of hu24 replaced CDRs

[0439] Example 4. Construction of chimeric antibodies

[0440] The variable region sequences of the murine anti-GPRC5D antibodies mAb1 and mAb24 were combined with the heavy chain constant region set forth in SEQ ID NO:45 and the light chain constant region set forth in SEQ ID NO:46, respectively, to obtain chimeric antibodies Chi-1 and Chi-24. Exemplarily, Chi-1 represents a chimeric antibody comprising the murine heavy chain variable region, light chain variable region, and the aforementioned constant regions of mAb1, and so on. The sequences of the heavy chain constant region and light chain constant region of the chimeric antibodies are as follows:

[0441] >Heavy chain constant region sequence (SEQ ID NO: 45):

[0442] >CL (SEQ ID NO: 46):

[0443] The heavy chain sequence of Chi-1 (SEQ ID NO: 47):

[0444] Light chain sequence of Chi-1 (SEQ ID NO: 48):

[0445] The heavy chain sequence of Chi-24 (SEQ ID NO: 49):

[0446] The light chain sequence of Chi-24 (SEQ ID NO: 50):

[0447] Example 5. Preparation and identification of anti-GPRC5D / CD3 bispecific antibodies

[0448] The CD3 binding molecules disclosed herein can 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 embodiments disclosed herein employ S107E.

[0449] Table 7. CDRs of S107E

[0450] The sequence of the S107E variable region is as follows:

[0451] >S107E-VH (SEQ ID NO: 57):

[0452] >S107E-VL (SEQ ID NO: 58):

[0453] The humanized anti-GPRC5D antibody variable region was combined with the variable region of the anti-CD3 antibody S107E, IgG1 (CH1), obscurin, titin, and the IgG1 mutant IgG1 (AA, L234A / L235A), respectively, to form two different bispecific antibodies, namely Formula 2-OG4S and Formula 4-OG4S.

[0454] Formula 2-0G4S (i.e., F2-0G4S) is an asymmetric molecule consisting of four chains:

[0455] Chain 1: VH (anti-GPRC5D)-IgG1 (CH1)-IgG1Fc (Knob);

[0456] Chain 2: VL (anti-GPRC5D)-CL;

[0457] Chain 3 (Ob(A0B2)-Hole): VH(S107E)-Ob(A0B2)-IgG1Fc(Hole);

[0458] Chain 4 (VL-Titin(A0B2)): VL(S107E)-Titin(A0B2), a schematic diagram of which is shown in FIG1 (OB stands for Obscurin).

[0459] in:

[0460] >Ob(A0B2)-Hole(SEQ ID NO: 59): Note: CD3 binding domain CDRs obtained according to Kabat numbering rules are The heavy chain constant region is marked with "italics", and Obscurin (A0B2) is marked with Markers, mutation sites (L234A, L235A, Y349C, T366S, L368A, Y407V) were used mark.

[0461] >VL-Titin(A0B2)(SEQ ID NO: 60): Note: CD3 binding domain CDRs obtained according to Kabat numbering rules are Marker, Titin (A0B2) mark.

[0462] Formula 4-0G4S (i.e., F4-0G4S) is an asymmetric molecule consisting of four chains:

[0463] Chain 1 (Ob(A0B2)-Knob): VH(S107E)-Ob(A0B2)-IgG1Fc(Knob);

[0464] Chain 2 (VL-Titin(A0B2)): VL(S107E)-Titin(A0B2);

[0465] Chain 3: VH (anti-GPRC5D)-IgG1 (CH1)-IgG1 Fc (Hole);

[0466] Chain 4: VL (anti-GPRC5D)-CL;

[0467] Its schematic diagram is shown in Figure 2 (OB stands for Obscurin).

[0468] >Ob(A0B2)-Knob(SEQ ID NO: 61): Note: CD3 binding domain CDRs obtained according to Kabat numbering rules are The heavy chain constant region is marked with "italics", and Obscurin (A0B2) is marked with Marker, mutation sites (L234A, L235A, S354C, T366W) are marked with mark.

[0469] >VL-Titin(A0B2)(SEQ ID NO: 60): Note: CD3 binding domain CDRs obtained according to Kabat numbering rules are Marker, Titin (A0B2) mark.

[0470] in:

[0471] >IgG1(CH1) (SEQ ID NO: 62):

[0472] >IgG1Fc(Knob)(SEQ ID NO: 63):

[0473] >IgG1Fc(Hole)(SEQ ID NO: 64):

[0474] >Obscurin(A0B2)(SEQ ID NO:83):

[0475] >Titin(A0B2)(SEQ ID NO:84):

[0476] Table 8. Bispecific antibodies of the present disclosure

[0477] The specific amino acid sequence is as follows:

[0478] Chain 1 of F2-0G4S-1 (SEQ ID NO: 65):

[0479] Chain 2 of F2-0G4S-1 (SEQ ID NO: 66):

[0480] Chain 3 of F2-0G4S-1 (SEQ ID NO: 59)

[0481] Chain 4 of F2-0G4S-1 (SEQ ID NO: 60)

[0482] Chain 1 of F4-0G4S-1 (SEQ ID NO: 61)

[0483] Chain 2 of F4-0G4S-1 (SEQ ID NO: 60)

[0484] Chain 3 of F4-0G4S-1 (SEQ ID NO: 67):

[0485] Chain 4 of F4-0G4S-1 (SEQ ID NO: 68):

[0486] The positive control molecule JNJ-64407564 used in this disclosure has the following sequence (derived from WO2018017786):

[0487] >Heavy chain (anti-GPRC5D) (SEQ ID NO: 69):

[0488] >Light chain (anti-GPRC5D) (SEQ ID NO: 70):

[0489] >Heavy chain (anti-CD3E) (SEQ ID NO: 71):

[0490] >Light chain (anti-CD3E) (SEQ ID NO: 72):

[0491] >Heavy chain variable region of the humanized anti-GPRC5D antibody in JNJ-64407564 (SEQ ID NO: 73):

[0492] >Light chain variable region of the humanized anti-GPRC5D antibody in JNJ-64407564 (SEQ ID NO: 74):

[0493] >Heavy chain of JNJ7564-huIgG1 (SEQ ID NO: 75):

[0494] >Light chain of JNJ7564-huIgG1 (SEQ ID NO: 76):

[0495] Test Example - Detection of a Bispecific Antibody that Binds Specifically to GPRC5D and CD3

[0496] Test Example 1. FACS determination of the affinity of GPRC5D / CD3 bispecific antibody for GPRC5D

[0497] To test the binding ability of the disclosed GPRC5D / CD3 bispecific antibody to GPRC5D, this test example used flow cytometry to detect the binding of the GPRC5D / CD3 bispecific antibody to the human multiple myeloma cell line MM.1R ( The binding capacity of CRL-2975) and the stable transgenic cell line CHOK1-cynoGPRC5D expressing cynomolgus macaque GPRC5D was tested as follows: the above cells were cultured in complete medium with 10% FBS in a 37°C, 5% CO2 incubator for 2 days, and the number of cells per well was 1×10 5 Add cells to the cell plate, centrifuge at 300g for 5 minutes, and wash once with 1% BSA. Dilute the antibody serially to 8 concentrations, add 100μL to each well of the cell plate, incubate at 4℃ for 1 hour, wash once with 1% BSA, add 100μL FITC-Goat Anti-human IgG Fc fluorescent secondary antibody dilution (1:400) to each well, and incubate at 4℃ for 1 hour. Wash the plate three times with 1% BSA, add 100μL PBS to each well and read the plate. EC of each antibody binding to the corresponding cells 50 (nM) are shown in the table below.

[0498] Table 9. Determination of the ability of chimeric antibodies to bind to GPRC5D Note: JNJ7564-huIgG1 is an antibody obtained by combining the variable region sequence of the humanized anti-GPRC5D antibody in JNJ-64407564 with the heavy chain constant region shown in SEQ ID NO: 45 and the light chain constant region shown in SEQ ID NO: 46; "-" indicates no binding was detected.

[0499] The results showed that the chimeric GPRC5D antibody disclosed herein had excellent binding to membrane-surface GPRC5D and strong cross-binding activity with cynoGPRC5D. JNJ-64407564 exhibited weaker binding to human GPRC5D than the antibody disclosed herein, primarily as evidenced by a very low binding response and very weak, almost undetectable, affinity for cynoGPRC5D.

[0500] Test Example 2. ELISA determination of the binding epitope between GPRC5D / CD3 bispecific antibody and GPRC5D

[0501] To determine the binding epitope of the disclosed GPRC5D / CD3 bispecific antibody to GPRC5D, four extracellular domain peptides of human GPRC5D were synthesized: the amino-terminal peptide (N-terminal peptide), the E2 peptide, the E3 peptide, and the E4 peptide (synthesized by GenScript). The peptides were biotinylated and prepared in two forms: lacking and containing intramolecular disulfide bonds. The sequences are shown in Table 10. Steptavidin (Sigma, SA101) was coated onto an Elisa plate (Corning 3590), incubated at 37°C for 2 hours, and then washed. Nonspecific 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 in PBS containing 1% BSA was added to each well. The plate was incubated at 37°C for 1 hour, and then washed. 100 μL of GPRC5D / CD3 antibody diluted to 0.003 nM-300 nM in 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. The plate was incubated at 37°C for 1 hour, and then washed. 100 μL of TMB (KPL, 5120-0077) was added to each well. After color development for 5 minutes, 1 M H2SO4 was added to terminate the color development, and the OD was measured. 450 The specific results are shown in Table 11.

[0502] Table 10. Four extracellular domain peptide sequences of human GPRC5D

[0503] Table 11. Determination of the ability of F2-0G4S-1 and F4-0G4S-1 to bind to the four extracellular domain peptides of GPRC5D Note: “-” indicates no binding was detected.

[0504] The results showed that the GPRC5D / CD3 bispecific antibodies F2-0G4S-1 and F4-0G4S-1 disclosed herein bound to the amino-terminal peptide sequence of human GPRC5D, regardless of whether the peptides formed intramolecular disulfide bonds. Furthermore, all tested antibodies did not bind to the E2, E3, and E4 peptides. These results indicate that the binding epitope of the GPRC5D / CD3 bispecific antibodies F2-0G4S-1 and F4-0G4S-1 disclosed herein to GPRC5D is located in the amino-terminal region of human GPRC5D.

[0505] Test Example 3. FACS determination of cross-binding between GPRC5D / CD3 bispecific antibody and GPCRA and GPCRB family members

[0506] To test whether the disclosed GPRC5D / CD3 bispecific antibody cross-binds with GPCRA and GPCRB family members, this test example used flow cytometry to detect the binding ability of the GPRC5D / CD3 bispecific antibody to CHOK1-human CCR8 cells expressing human CCR8 (GPCR A family) (constructed with reference to Example 1) and CHOK1-human GIPR cells expressing human GIPR (GPCR B family) (constructed with reference to Example 1). Specifically, the above cells were cultured in DMEF / 12K medium containing 10% FBS and placed in a 37°C, 5% CO2 incubator for 2 days. The number of cells per well was 1×10 5 Cells were added to the cell plate, centrifuged at 300g for 5 minutes, and washed once with 1% BSA. Antibody was serially diluted to eight concentrations, and 100 μL was added to each well of the cell plate. The plate was incubated at 4°C for 1 hour. The plate was washed once with 1% BSA, and 100 μL of FITC-Goat Anti-human IgG Fc fluorescent secondary antibody diluted in buffer (1:400) was added to each well. The plate was incubated at 4°C for 1 hour. The plate was washed three times with 1% BSA, and 100 μL of PBS was added to each well for reading.

[0507] >Human CCR8 antigen (SEQ ID NO: 81):

[0508] >Human GIPR antigen (SEQ ID NO: 82):

[0509] The results showed that the GPRC5D / CD3 bispecific antibodies F2-0G4S-1 and F4-0G4S-1 disclosed herein did not bind to human CCR8 and human GIPR, indicating their good specificity.

[0510] Test Example 4. In vitro cytotoxic activity of the bispecific antibodies disclosed herein

[0511] This test example studies the cytotoxic activity of the bispecific antibody disclosed herein as a T cell engagement molecule against tumor cells. The cell line H929 expressing GPRC5D and the Daudi cell line not expressing GPRC5D were used as target cells to detect the target-specific cytotoxic activity of the bispecific antibody disclosed herein. Fresh PBMC (purchased from Miaoshun Biotechnology Co., Ltd.) was centrifuged at 300g for 10 minutes, the supernatant was discarded, and the suspension was resuspended in 1640+10% FBS (liquid A) containing 200U IL2 to form a 2E6 / mL cell suspension, and placed in a culture flask for overnight culture. After overnight activation, the PBMCs were collected and centrifuged and resuspended in 1640+4% FBS without phenol red. After re-centrifugation, they were resuspended and counted, and the cell number was adjusted to 6.25E5 cells / mL. 60μL was added to each well (the number of PBMC cells in each well was 37,500). Target cells NCI-H929 (ATCC, CRL-9068) were collected and centrifuged at 1000 rpm for 3 minutes. The cell count was adjusted to 1.25E5 cells / mL and 60 μL was added to each well (7500 target cells per well), ensuring an E:T ratio of 5:1. The antibody was diluted in phenol red-free complete medium with a starting concentration of 500 nM (5× final concentration). Nine 10-fold dilutions were made and 30 μL was added to each well. The cells were then incubated at 37°C, 5% CO2 for 48 hours. Before detection, 10 μL of medium was aspirated from two wells containing only target cells, and 10 μL of Lysis Solution (10×) was added. After lysis for 45 minutes, the culture plate was removed and centrifuged at 1000 rpm for 3 minutes. 50 μL of the supernatant was aspirated and transferred to a new 96-well plate (#3590). 50 μL of the dissolved CytoTox was added at a 1:1 ratio. Reagent, incubate at room temperature for 0.5 h, then add 50 μL of stop solution, and measure absorbance (490 nm) using FlexStation 3 (Molecular Devices). Specific results are shown in the table below.

[0512] Table 12. Cytotoxic activity of antibodies against NCI-H929 Note: EC 50 Ratio (relative to JNJ-64407564) = EC 50 (antibody to be tested) / EC 50 (JNJ-64407564).

[0513] The results showed that the GPRC5D / CD3 bispecific antibody disclosed herein had stronger NCI-H929 cytotoxic activity than JNJ-64407564; moreover, the cytotoxic activity of the GPRC5D / CD3 bispecific antibodies F2-0G4S-1 and F4-0G4S-1 disclosed herein was specific to the GPRC5D target, and no killing activity was detected in the Daudi cell line without GPRC5D expression, suggesting that the GPRC5D / CD3 bispecific antibody disclosed herein has better safety.

[0514] Test Example 5. Cytokine Release Level of GPRC5D / CD3 Bispecific Antibody

[0515] CD3T cell engagement molecules can cause cytokine storms. Therefore, when developing CD3T cell engagement molecules, it is necessary to keep cytokines, especially IL6, a factor that has nothing to do with drug efficacy but can cause side effects, at a low level. This test case studies the release levels of cytokines IFNγ and IL6 of the bispecific antibodies disclosed herein. Fresh PBMC (purchased from Miaoshun Biological Company) was centrifuged at 300g for 10 minutes, the supernatant was discarded, and the cells were resuspended into 2E6 / mL cell suspension with 1640+10% FBS (A solution) containing 200U IL2 and placed in a culture flask for overnight culture. PBMC activated overnight were collected and centrifuged and resuspended with 1640+4% FBS without phenol red. After re-centrifugation, they were resuspended and counted, and the cell number was adjusted to 6.25E5 cells / mL. 60μL was added to each well (the number of PBMC cells in each well was 37,500). Target cells were harvested from NCI-H929 (ATCC, CRL-9068) or RPMI-8226 (ATCC, CRM-CCL-155) and centrifuged at 1000 rpm for 3 minutes. The cell count was adjusted to 1.25E5 cells / mL, and 60 μL was added to each well (7500 target cells per well), ensuring an E:T ratio of 5:1. Antibodies were diluted in phenol red-free complete medium at a starting concentration of 500 nM (5× final concentration). Nine 10-fold dilutions were made, and 30 μL was added to each well. Cells were then incubated at 37°C, 5% CO2 for 48 hours before use.

[0516] 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 universal specimen diluent, and the IL6 standard was also diluted 6 times. The diluted samples or different concentrations of standards (100 μL / well) were added to the detection plate, and the reaction wells were sealed with sealing tape. After incubation at 37°C for 90 minutes, the plate was washed 5 times, and the biotinylated antibody working solution (100 μL / well) was added. The reaction wells were sealed with new sealing tape. After incubation at 37°C for 60 minutes, the plate was washed 5 times, and the enzyme conjugate working solution (100 μL / well) was added. The reaction wells were sealed with new sealing tape. After incubation at 37°C for 30 minutes, the plate was washed 5 times, and 100 μL / well of colorimetric substrate (TMB) was added. Incubated at 37°C for 8 minutes in the dark, 100 μL / well of reaction stop solution was added, and the OD was measured immediately after mixing. 450 value (within 3 minutes).

[0517] IFNγ was detected using the HTRF method. The above cell samples were centrifuged at 1000rpm for 3 minutes, 50μL of cell supernatant was collected, the detection kit was balanced to room temperature, and the two detection antibodies in the kit were diluted with detection buffer (20-fold dilution). 16μL of the 20-fold diluted sample and IFNγ standard were placed in a 384-well plate, and 4μL of the diluted corresponding detection antibody was added; the sealing material was attached, shaken to mix, centrifuged at 1000rpm for 1 minute, incubated at room temperature overnight, centrifuged at 1000rpm for 1 minute, and the sealing material was removed. The absorbance values ​​at 665nm and 620nm were read using a PHERAstar multifunctional microplate reader, and the data were processed and analyzed using Graphpad Prism 5. The specific results are shown in the table below.

[0518] Table 13-1. IL-6 release in RPMI-8226 cytotoxicity assay Note: “-” indicates that the result was below the detection limit and not detected, the same below.

[0519] Table 13-2. IL-6 release in RPMI-8226 cytotoxicity assay

[0520] Table 13-3. IFNγ Release in RPMI-8226 Cytotoxicity Assay

[0521] Table 13-4. IFNγ Release in RPMI-8226 Cytotoxicity Assay

[0522] Table 13-5. IL-6 release in NCI-H929 cytotoxicity assay

[0523] Table 13-6. Release of IL-6 in NCI-H929 Cytotoxicity Assay

[0524] The results showed that the GPRC5D / CD3 bispecific antibody disclosed herein released lower levels of IL6 and IFNγ than JNJ-64407564, with IL6, in particular, being almost undetectable, suggesting that the GPRC5D / CD3 bispecific antibody disclosed herein has a better safety profile.

[0525] In vivo biological evaluation

[0526] Test Example 6. Efficacy of GPRC5D / CD3 bispecific antibody in NCIH929 subcutaneous transplant tumor model

[0527] The present disclosure uses a human myeloma NCI-H929 cell human PBMC NOG mouse xenograft tumor model to evaluate the anti-tumor activity of the GPRC5D / CD3 bispecific antibody.

[0528] NOG mice, female, 8-10 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0529] The human multiple myeloma cell line NCI-H929 was purchased from Shanghai Mingjin Biotechnology Co., Ltd.

[0530] NCI-H929 cells were added to RPMI-1640 medium containing 10% FBS and 0.05 mM 2-mercaptoethanol and cultured in a humidified incubator at 37°C with 5% CO2. NCI-H929 cells in the logarithmic growth phase were collected and resuspended in RPMI-1640 medium containing 50% Matrigel to adjust the cell density to 2 × 10 7 Under sterile conditions, 0.1 mL of cell suspension was inoculated subcutaneously on the right side of the mouse back at a concentration of 2 × 10 6 / 0.1mL / mouse. One day after tumor cell inoculation, hPBMCs frozen in liquid nitrogen were revived and cultured in PRMI-1640 medium containing 10% HIFBS (FBS, inactivated at 56°C for 30 minutes). The cells were incubated in a 37°C incubator with 5% CO2 for 6 hours. After incubation, hPBMCs were collected and resuspended in PBS buffer to adjust the cell concentration to 2.5×10 7 Under sterile conditions, 0.2 mL of cell suspension was injected intraperitoneally into mice at an injection concentration of 5 × 10 6 / 0.2mL / mouse. When the average tumor volume reaches 100mm 3Around 24 hours post-treatment, animals were randomly divided into groups based on tumor volume, ensuring that the difference in tumor volume between groups was less than 10% of the mean. The day of grouping was designated Day 0, and dosing began according to animal body weight. Immediately after grouping, dosing was performed intraperitoneally once weekly for two consecutive doses (IP, qW x 2), and tumor volume was recorded.

[0531] Tumor inhibition rate (%) = 1-T / C (%).

[0532] T / C(%)=(T-T0) / (C-C0)×100%.

[0533] Tumor volume (V) = 1 / 2 × a × b 2 , where a and b represent length and width respectively.

[0534] T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0535] The specific results are shown in the table below, where the tumor inhibition rates calculated in the table are all the tumor inhibition rates on the last day.

[0536] Table 14. Efficacy of F4-0G4S-1 and F2-0G4S-1 in NCIH929 subcutaneous transplant tumor model

[0537] Experimental results demonstrated that the disclosed GPRC5D / CD3 bispecific antibodies F2-0G4S-1 and F4-0G4S-1 demonstrated greater in vivo tumor growth inhibition activity than JNJ-64407564 at the indicated dosages. No drug-related animal deaths or other significant drug-related side effects were observed during the experiments.

[0538] Preparation Example - Bispecific Antibody Preparation Specifically Binding to GPRC5D and CD3

[0539] SEC size exclusion chromatography:

[0540] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0541] SEC% (SEC monomer content percentage) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, A total is the sum of all peak areas). ΔSEC% = SEC% of the preparation before the stability experiment - SEC% of the preparation after the stability experiment.

[0542] SEC measurement instrument: Agilent HPLC 1260;

[0543] Column: Waters, SEC 3.5μm 7.8×300mm Column

[0544] NR-CE capillary gel electrophoresis:

[0545] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0546] NR-CE% (NR-CE main peak content percentage) = Amainpeak / Atotal × 100% (Amainpeak is the peak area of ​​the main peak in the sample, and Atotal is the sum of all peak areas). ΔNR-CE% = NR-CE% of the preparation before the stability experiment - NR-CE% of the preparation after the stability experiment.

[0547] CE measurement instrument: Beckman capillary electrophoresis instrument, model PA800plus

[0548] IEC ion exchange chromatography:

[0549] A chromatographic method that uses ion exchange resin or chemically bonded ion exchanger as the stationary phase and utilizes the difference in ion exchange capacity or selectivity coefficient of the separated components to achieve separation.

[0550] IEC% (IEC neutral peak content percentage) = A neutral peak area / A total area × 100% (A total area is the sum of the acidic peak, neutral peak and basic peak areas). ΔIEC% = IEC% of the preparation before the stability test - IEC% of the preparation after the stability test.

[0551] IEC measurement instrument: Agilent HPLC 1260.

[0552] iCIEF whole-column imaging capillary isoelectric focusing electrophoresis:

[0553] A technique for separating proteins based on their isoelectric points (pI).

[0554] iCIEF% (iCIEF neutral peak content percentage) = A neutral peak area / A total area × 100% (A total area is the sum of the acidic peak, neutral peak, and basic peak areas). ΔiCIEF% = iCIEF% of the formulation before the stability experiment - iCIEF% of the formulation after the stability experiment.

[0555] Instrument used for iCIEF determination: simple protein, model Muarice.

[0556] ELSD anion exchange and reversed phase adsorption chromatography:

[0557] A method for quantifying polysorbate 80 is proposed, which separates solutes based on the principles of anion exchange and reverse adsorption and analyzes them using charged aerosol detection technology.

[0558] PS80 degradation rate (%) = (PS80 concentration of the preparation before the stability experiment, mg / mL - PS80 concentration of the preparation after the stability experiment, mg / mL) / PS80 concentration of the preparation before the stability experiment, mg / mL.

[0559] ELSD measurement instrument: Agilent HPLC 1260.

[0560] Osmolality determination:

[0561] The freezing point method for determining osmotic pressure is based on the fact that the freezing point depression value is directly proportional to the molar concentration of the solution. A highly sensitive temperature sensing element is used to determine the freezing point of the solution and convert the electrical quantity into osmotic pressure.

[0562] Instrument for osmotic pressure measurement: Loser, model OM815.

[0563] protein

[0564] The protein used in the following formulation examples is the GPRC5D-CD3 bispecific antibody F4-0G4S-1 (hereinafter referred to as "protein") described above.

[0565] The protein concentration was determined using a UV-visible spectrophotometer, model: Nano Drop oneC, with an optical path length of 1 mm.

[0566] Formulation Example 1. pH and buffer system screening

[0567] A formulation containing 100 mg / mL protein, 80 mg / mL sucrose, and 0.4 mg / mL polysorbate 80 (PS80) was prepared using the buffer system shown in Table 15-1. Forced degradation studies (2 weeks at 40°C) were conducted to investigate the effects of different pH values ​​and buffer systems on protein stability, using SEC, NR-CE, and iCIEF as evaluation criteria.

[0568] The results are shown in Table 15-1. SEC data showed that the monomer purity of the preparation with a pH of 5.5-6.0 after 2 weeks at 40°C was superior to that of the other groups. NR-CE data showed that the ΔNR-CE values ​​fluctuated within the range of 0.4-1.4%, with minimal differences between the groups. iCIEF data showed that the neutral peak purity of the preparation containing AA, His, and CA was superior to that of the other groups.

[0569] Table 15-1 pH and buffer system screening results-1 Note: AA stands for acetic acid-sodium acetate; His-HCl stands for histidine-histidine hydrochloride; SA stands for succinic acid-sodium succinate; CA stands for citric acid-sodium citrate; PB stands for disodium hydrogen phosphate-sodium dihydrogen phosphate; 40℃ W2: 40℃ for 2 weeks, the same below.

[0570] The samples were subjected to forced degradation studies (25°C, 300 rpm shaking for 7 days), and the effects of different pH and buffer systems on protein stability were investigated using appearance as an evaluation indicator.

[0571] The results are shown in Table 15-2. Appearance showed that after 7 days of shaking, the preparations containing SA and CA had fine particles on their surface.

[0572] Table 15-2 pH and buffer system screening results-2 Note: Shaking D7: Shake for 7 days, same below.

[0573] In summary, the data from the forced degradation study (40°C for 2 weeks; 25°C, 300 rpm shaking for 7 days) showed that the AA, His-HCl system, and pH 5.5-6.0 formulations were superior to the other groups.

[0574] Formulation Example 2. Buffer System Ionic Strength Screening

[0575] Formulations containing 100 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL PS80, and 0.1 mg / mL edetate disodium (CAS No. 6381-92-6, purchased from SIGMA) were prepared using the buffer systems of varying ionic strengths shown in Table 16. Forced degradation studies (40°C for 4 weeks) were conducted to investigate the effects of different buffer ionic strengths on protein stability, using appearance, SEC, NR-CE, and IEC as evaluation criteria.

[0576] The results are shown in Table 16. The data showed that after 4 weeks at 40°C, there was no significant difference between the formulations containing different buffer system ionic strengths.

[0577] Table 16 Buffer system ionic strength screening results

[0578] Formulation Example 3. High Protein Concentration Screening

[0579] Formulations containing 80 mg / mL sucrose, 0.4 mg / mL PS80, and the protein concentrations shown in Table 17 were prepared using 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks) and 2-8°C for 6 months were performed on the samples. Due to the increased risk of SEC aggregates at high protein concentrations, SEC was used as an evaluation metric to examine the effect of different protein concentrations on protein stability.

[0580] The results are shown in Table 17. The data showed that with the increase of protein concentration, ΔSEC had a slight increasing trend, but the difference between the groups was small and had good stability.

[0581] Table 17 Protein concentration screening results Note: 40℃ W4: 40℃ for 4 weeks; 2-8℃ M6: 2-8℃ for 6 months. Same below.

[0582] Formulation Example 4. Low Protein Concentration Screening

[0583] Formulations containing 80 mg / mL sucrose, 0.4 mg / mL PS80, 0.1 mg / mL edetate disodium, and the protein concentrations shown in Table 18 were prepared using 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks) were performed on the samples. Because the risk of SEC aggregates is low at low protein concentrations, the effects of different protein concentrations on protein stability were evaluated using appearance, NR-CE, and IEC as evaluation indicators.

[0584] The results are shown in Table 18. The data showed that after 4 weeks at 40°C, there was little difference in NR-CE and IEC between the formulations containing different protein concentrations.

[0585] Table 18 Protein concentration screening results

[0586] Formulation Example 5. Protein Concentration Confirmation

[0587] Formulations containing 80 mg / mL sucrose, 0.4 mg / mL PS80, 0.1 mg / mL edetate disodium, and the protein concentrations shown in Table 19 were prepared using 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks) and long-term stability studies at 2-8°C were performed on the samples. The effects of different protein concentrations on protein stability were evaluated using appearance, SEC, NR-CE, and IEC as evaluation criteria.

[0588] The results are shown in Table 19. The data show that after 4 weeks at 40°C, there was minimal difference in purity between groups. After 3 months at 2-8°C, there was no significant change in purity in the 2 mg / mL and 60 mg / mL protein concentration groups. After 12 months at 2-8°C, there was no significant change in NR-CE in the 100 mg / mL protein concentration group, while SEC and IEC values ​​decreased slightly. All samples containing different protein concentrations showed good stability.

[0589] Table 19 Protein concentration screening results Note: 2-8℃ M12: 12 months at 2-8℃. 2-8℃ M3: 3 months at 2-8℃.

[0590] Preparation Example 6. Excipient Screening

[0591] Formulations of 100 mg / mL protein, with or without the addition of edetate disodium as shown in Table 20-1, 80 mg / mL sucrose, and 0.4 mg / mL PS80 were prepared using 20 mM His-HCl buffer (pH 5.5).

[0592] Table 20-1 Screening table for different excipients

[0593] A forced degradation study (40°C for 4 weeks) was conducted on the samples, using the degradation rate of PS80 as an evaluation indicator to examine the effects of different excipients on the stability of the formulation. The results are shown in Table 20-2. The PS80 degradation rate data showed that after 4 weeks at 40°C, the PS80 degradation rate of the formulation containing edetate disodium was significantly lower than that of the formulation without edetate disodium.

[0594] Table 20-2 Excipient screening results

[0595] Preparation Example 7. Edetate Disodium Concentration Screening

[0596] Formulations containing 100 mg / mL protein, various concentrations of edetate disodium, 80 mg / mL sucrose, and 0.4 mg / mL PS80 were prepared in 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks) were conducted to investigate the effects of various excipients on formulation stability, using SEC, NR-CE, IEC, and PS80 degradation rates as evaluation indicators.

[0597] The results are shown in Table 21. The data show that after being placed at 40°C for 4 weeks, there is no significant difference between preparations containing different concentrations of edetate disodium.

[0598] Table 21 Edetate disodium concentration screening results

[0599] Formulation Example 8. Sugar Concentration Screening

[0600] Sugar is a commonly used excipient in biological drugs. This disclosure uses sucrose as an osmotic pressure regulator and stabilizer for prescription screening, which can meet the formulation requirements.

[0601] Formulations containing 100 mg / mL protein, 0.4 mg / mL PS80, 0.1 mg / mL edetate disodium, and varying sucrose concentrations as shown in Table 22 were prepared using 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks) were performed to investigate the effects of varying sucrose concentrations on protein stability, using appearance, SEC, NR-CE, and IEC as evaluation criteria.

[0602] The results are shown in Table 22. The data showed that after 4 weeks at 40°C, there was no significant difference between the formulations containing different concentrations of sucrose.

[0603] Table 22 sucrose concentration screening results

[0604] Formulation Example 9. Screening of surfactant types

[0605] Formulations containing 100 mg / mL protein, 80 mg / mL sucrose, and the various surfactants listed in Table 23 were prepared in 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks) were performed to investigate the effects of various surfactants on protein stability using SEC, NR-CE, and IEC.

[0606] The results are shown in Table 23. The data showed that after 4 weeks at 40°C, there was no significant difference between the formulations containing different types of surfactants.

[0607] Table 23 Surfactant type screening results

[0608] Formulation Example 10 Surfactant Concentration Screening-1

[0609] Formulations containing 100 mg / mL protein, 80 mg / mL sucrose, 0.1 mg / mL edetate disodium, and various PS80 concentrations as shown in Table 24 were prepared using 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks, shaken for 7 days) were performed on the samples to investigate the effects of different surfactant concentrations on formulation stability, using SEC as an evaluation metric.

[0610] The results are shown in Table 24. Appearance and SEC data showed that after being placed at 40°C for 4 weeks and shaken for 7 days, there was no significant difference in the preparations containing different concentrations of PS80.

[0611] Table 24 Edetate disodium concentration screening results

[0612] Formulation Example 11. Surfactant Concentration Screening-2

[0613] Formulations containing 100 mg / mL protein, 80 mg / mL sucrose, 0.1 mg / mL edetate disodium, and various PS80 concentrations as shown in Table 25 were prepared using 20 mM His-HCl buffer (pH 5.5). Forced degradation studies (40°C for 4 weeks) were conducted to investigate the effects of different surfactant concentrations on protein stability, using appearance, SEC, NR-CE, and IEC as evaluation criteria.

[0614] The results are shown in Table 25. The data showed that after 4 weeks at 40°C, there was no significant difference between the formulations containing different concentrations of PS80.

[0615] Table 25 Surfactant screening results

Claims

1. A pharmaceutical composition comprising a bispecific antibody that specifically binds to GPRC5D and CD3 and a buffer, wherein: The bispecific antibody that specifically binds to GPRC5D and CD3 comprises an antigen binding module 1 that specifically binds to GPRC5D and an antigen binding module 2 that specifically binds to CD3, wherein the antigen binding module 1 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 2 that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL; in, (i) the GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, 14, 41, 42 or 44, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16, or The GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, 6 or 30, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 27, 8, 26, 28 or 29, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10; and (ii) the CD3-VH comprises: a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 53, and the CD3-VL comprises: a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 56; The buffer is a histidine buffer, an acetate buffer, a citrate buffer, a succinate buffer or a phosphate buffer; Preferably, the buffer is histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; More preferably, the buffer is histidine-histidine hydrochloride buffer.

2. The pharmaceutical composition according to claim 1, wherein the pH of the pharmaceutical composition is 5.0 to 7.0; preferably, the pH of the pharmaceutical composition is 5.0 to 6.5; further preferably, the pH of the pharmaceutical composition is 5.0 to 6.0; more preferably, the pH of the pharmaceutical composition is 5.5 to 6.0; most preferably, the pH of the pharmaceutical composition is about 5.

5.

3. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 1 mg / mL to 250 mg / mL; Preferably, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 1 mg / mL to 200 mg / mL; Further preferably, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 1 mg / mL to 150 mg / mL; More preferably, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 60 mg / mL to 150 mg / mL; More preferably, the concentration of the bispecific antibody that specifically binds to GPRC5D and CD3 is 80 mg / mL to 120 mg / mL; Most preferably, the concentration of the bispecific antibody that specifically binds GPRC5D and CD3 is about 2 mg / mL, about 60 mg / mL, or about 100 mg / mL.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises a surfactant; preferably, the surfactant is polysorbate or poloxamer; more preferably, the surfactant is polysorbate 80 or poloxamer 188; most preferably, the surfactant is polysorbate 80.

5. The pharmaceutical composition according to claim 4, wherein the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL; preferably, the concentration of the surfactant is 0.1 mg / mL to 0.8 mg / mL; further preferably, the concentration of the surfactant is 0.2 mg / mL to 0.6 mg / mL; more preferably, the concentration of the surfactant is 0.32 mg / mL to 0.48 mg / mL; most preferably, the concentration of the surfactant is about 0.4 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition comprises sugar; preferably, the sugar is sucrose, trehalose, mannitol or sorbitol; more preferably, the sugar is sucrose.

7. The pharmaceutical composition of claim 6, wherein the concentration of the sugar is 10 mg / mL to 100 mg / mL; preferably, the concentration of the sugar is 30 mg / mL to 100 mg / mL; more preferably, the concentration of the sugar is 64 mg / mL to 96 mg / mL; most preferably, the concentration of the sugar is about 80 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the concentration of the buffer is 5 mM to 100 mM; preferably, the concentration of the buffer is 5 mM to 50 mM; further preferably, the concentration of the buffer is 5 mM to 30 mM; more preferably, the concentration of the buffer is 8 mM to 20 mM; most preferably, the concentration of the buffer is about 14 mM or about 20 mM.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition further comprises an excipient; preferably, the excipient is disodium ethylenediaminetetraacetic acid or a hydrate thereof, arginine hydrochloride, glycine, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride or calcium chloride; more preferably, the excipient is disodium ethylenediaminetetraacetic acid dihydrate.

10. The pharmaceutical composition according to claim 9, wherein the concentration of the excipient is 0.01 mg / mL to 1 mg / mL; preferably, the concentration of the excipient is 0.01 mg / mL to 0.2 mg / mL; more preferably, the concentration of the excipient is 0.08 mg / mL to 0.12 mg / mL; most preferably, the concentration of the excipient is about 0.1 mg / mL.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the bispecific antibody that specifically binds to GPRC5D and CD3, (i) the GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 43, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16, or The GPRC5D-VH has: a GPRC5D-HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, a GPRC5D-HCDR2 comprising the amino acid sequence of SEQ ID NO: 31, a GPRC5D-HCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and the GPRC5D-VL has: a GPRC5D-LCDR1 comprising the amino acid sequence of SEQ ID NO: 27, a GPRC5D-LCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a GPRC5D-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10; and (ii) the CD3-VH comprises: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 53; and the CD3-VL comprises: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:

56.

12. The pharmaceutical composition according to claim 11, wherein the bispecific antibody that specifically binds to GPRC5D and CD3: (i) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 34, 32 or 33, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 34, 32 or 33, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 39, 35, 36, 37, 38, 40, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39, 35, 36, 37, 38, 40; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3, and the GPRC5D-VL comprises SEQ ID NO: The amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4; and the CD3-VH comprises the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:58; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 19, 17 or 18, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 19, 17 or 18, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 23, 20, 21, 22, 24 or 25, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 23, 20, 21, 22, 24 or 25; or The GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2; and the CD3-VH comprises the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:58; Preferably, (i) 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; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 58; or (ii) the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 19, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 23; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 57, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:

58.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the antigen binding moiety that specifically binds to GPRC5D is Fab; the antigen binding moiety that specifically binds to CD3 is a replaced Fab; the replaced Fab is a Fab in which the original CH1 and CL are replaced by the Obscurin chain and the Titin chain, respectively; Preferably, the Obscurin chain comprises the amino acid sequence of SEQ ID NO:83, and the Titin chain comprises the amino acid sequence of SEQ ID NO:

84.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the bispecific antibody that specifically binds to GPRC5D and CD3 has a first chain as shown in SEQ ID NO: 61, a second chain as shown in SEQ ID NO: 60, a third chain as shown in SEQ ID NO: 67, and a fourth chain as shown in SEQ ID NO: 68; or It has a first strand as shown in SEQ ID NO:65, a second strand as shown in SEQ ID NO:66, a third strand as shown in SEQ ID NO:59 and a fourth strand as shown in SEQ ID NO:

60.

15. A pharmaceutical composition according to any one of claims 1 to 14, comprising the following components: (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 10 mg / mL to 100 mg / mL of sugar, (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt or its hydrate, and (e) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition is 5.0 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3, (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80, (c) 30 mg / mL to 100 mg / mL of sucrose, (d) 0.01 mg / mL to 1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and (e) 5 mM to 50 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0; Further preferably, the pharmaceutical composition comprises the following components: (a) 60 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to GPRC5D and CD3, (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80, (c) 64 mg / mL to 96 mg / mL of sucrose, (d) 0.01 mg / mL to 0.2 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and (e) 5 mM to 30 mM histidine-histidine hydrochloride buffer or acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) 80 mg / mL to 120 mg / mL of a bispecific antibody that specifically binds to GPRC5D and CD3, (b) 0.32 mg / mL to 0.48 mg / mL of polysorbate 80, (c) 64 mg / mL to 96 mg / mL of sucrose, (d) 0.08 mg / mL to 0.12 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and (e) 8 mM to 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5 to 6.0; Most preferably, the pharmaceutical composition comprises the following components: (a) about 2 mg / mL, or about 60 mg / mL, or about 100 mg / mL of a bispecific antibody that specifically binds GPRC5D and CD3, (b) about 0.4 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5; or (a) about 2 mg / mL, or about 60 mg / mL, or about 100 mg / mL of a bispecific antibody that specifically binds GPRC5D and CD3, (b) about 0.4 mg / mL of polysorbate 80, (c) about 80 mg / mL of sucrose, (d) about 0.1 mg / mL of ethylenediaminetetraacetic acid disodium salt dihydrate, and (e) about 14 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.

5.

16. A lyophilized preparation, which can form the pharmaceutical composition according to any one of claims 1 to 15 after being reconstituted.

17. A method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 15.

18. A lyophilized preparation obtained by the method of claim 17.

19. The pharmaceutical composition according to any one of claims 1 to 15, which is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation or an intramuscular injection preparation; preferably a subcutaneous injection preparation.

20. A method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 15, or the lyophilized preparation of claim 16 or 18; Preferably, the disease is selected from tumors, cancers and autoimmune diseases; More preferably, the disease is selected from 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, Waldenstrom's macroglobulinemia, plasma cell leukemia, light chain amyloidosis, precursor B-cell lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndrome, syndrome, chronic lymphocytic leukemia, B-cell malignancies, chronic myeloid leukemia, hairy cell leukemia, blastic plasmacytoid dendritic cell neoplasm, Hodgkin lymphoma, non-Hodgkin 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, kidney cancer, liver cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, and melanoma.

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