Pharmaceutical composition comprising bispecific antibody specifically binding DLL3 and CD3

Abstract

Provided is a pharmaceutical composition comprising a bispecific antibody specifically binding DLL3 and CD3. Specifically, provided is a pharmaceutical composition, comprising a bispecific antibody specifically binding DLL3 and CD3 and a buffering agent.

Description

A pharmaceutical composition comprising a bispecific antibody that specifically binds to DLL3 and CD3 Technical Field

[0001] The present disclosure belongs to the field of biotechnology pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to DLL3 and CD3. Background Art

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

[0003] Small cell lung cancer (SCLC) is a relatively malignant type of lung cancer, accounting for 10%-15% of all lung cancer cases. Small cell lung cancer tumors grow rapidly and are prone to metastasis, with a 5-year survival rate of less than 7%. Treatment options for small cell lung cancer are limited, primarily consisting of chemotherapy, such as platinum / etoposide combination chemotherapy. Small cell lung cancer patients respond well to chemotherapy initially, but are very prone to developing drug resistance and relapse. In recent years, immunotherapies, such as PD-L1 and PD1 antibodies, have shown some effectiveness in SCLC patients, but the efficacy is approximately 15%. Currently, no specific targeted therapy has been developed.

[0004] DLL3 is a ligand that inhibits Notch. Normally, DLL3 resides on the Golgi apparatus. In cancer cells (such as small cell lung cancer), DLL3 is expressed on the cell surface. DLL3 binds to Notch in a cis-acting manner, hindering cell-cell binding and Notch internalization in target cells, thereby inhibiting the Notch signaling pathway and promoting tumor cell growth. DLL3 is primarily expressed in neural or neuroendocrine tumors, including small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, small cell bladder cancer, glioblastoma multiforme, metastatic castration-resistant prostate cancer, and melanoma. DLL3 is particularly expressed in SCLC, with over 80% of SCLCs expressing DLL3, whereas normal lung cancer tissue and adjacent adjacent tissues are negative. This differential expression makes DLL3 a promising therapeutic target for the treatment of SCLC.

[0005] CD3 is a homotypic or heterotypic dimeric antigen expressed on T cells. Functional CD3 is formed by dimerization of two of four different chains: ε, ζ, δ, and γ. CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. CD3 binds to the T cell receptor complex (TCR) and is required for T cell activation. Therefore, the use of anti-CD3 antibodies that activate T cells has been proposed for the treatment of cancer. However, the administration of anti-CD3 antibodies may trigger T cell activation and related cytokine release. Excessive cytokine release leads to severe cytokine release syndrome (CRS), which is an important challenge in the clinical use of anti-CD3 antibodies. Summary of the Invention

[0006] The present disclosure provides a pharmaceutical composition of a bispecific antibody that specifically binds to DLL3 and CD3 and uses thereof.

[0007] In one aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds DLL3 and CD3 and a buffer.

[0008] In one aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to DLL3 and CD3 and a buffer, wherein:

[0009] The bispecific antibody that specifically binds to DLL3 and CD3 comprises a first chain having a structure shown in Formula I and a second chain having a structure shown in Formula II.

[0010] Formula I: [DLL3-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1],

[0011] Formula II: [CD3-VL]-[Linker 3]-[DLL3-VH]-[Linker 2]-[Fc2],

[0012] the DLL3-VH comprising: a DLL3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a DLL3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a DLL3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 16; and the DLL3-VL comprising: a DLL3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 17, a DLL3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a DLL3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and

[0013] The CD3-VH comprises: a CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 56, a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 57, and a CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; and the CD3-VL comprises: a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 59, a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 61;

[0014] Wherein: the structures represented by Formula I and Formula II are arranged from N-terminus to C-terminus; the linker 1, linker 2, and linker 3 are identical or different peptide linkers; the Fc1 and the Fc2 are Fc region structural sequences that can associate with each other using a knob-in-hole technique;

[0015] The buffer is a histidine buffer, an acetate buffer, a citrate buffer, a succinate buffer or a phosphate buffer.

[0016] In some embodiments, the aforementioned buffer is histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, or citric acid-sodium citrate buffer.

[0017] In some embodiments, the buffer as described in any of the preceding items is histidine-histidine hydrochloride buffer.

[0018] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the pH of the pharmaceutical composition is between 4.5 and 6.0; preferably, the pH of the pharmaceutical composition is between 4.5 and 5.5; more preferably, the pH of the pharmaceutical composition is between 4.8 and 5.2. In some embodiments, the pH of the pharmaceutical composition is about 4.5. In some embodiments, the pH of the pharmaceutical composition is 4.5. In some embodiments, the pH of the pharmaceutical composition is about 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.0.

[0019] In some embodiments, the pH of any of the pharmaceutical compositions is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, or any range between these values. When a value is mentioned in this disclosure, it should be understood that the value includes a margin of error. This margin of error is due to factors such as laboratory environment, personnel operation, instrumentation, methodology, and measurement error. Taking pH as an example, when a measured value is about 5.0, it should be understood that it includes a margin of error. As an example, when an industrial pH meter is used to measure a preparation, "about 5.0" means 5.0 ± 0.2 (i.e., a pH of 4.8 to 5.2).

[0020] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL to 250 mg / mL.

[0021] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is between 1 mg / mL and 150 mg / mL. In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is between 5 mg / mL and 150 mg / mL. In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is between 1 mg / mL and 120 mg / mL. In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is between 1 mg / mL and 100 mg / mL. In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is between 5 mg / mL and 100 mg / mL. In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is between 70 mg / mL and 70 mg / mL.

[0022] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 70 mg / mL to 150 mg / mL.

[0023] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 80 mg / mL to 120 mg / mL.

[0024] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 90 mg / mL to 110 mg / mL.

[0025] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL to 20 mg / mL.

[0026] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL to 10 mg / mL.

[0027] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 4 mg / mL to 6 mg / mL.

[0028] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 1 mg / mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 5 mg / mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 5 mg / mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 70 mg / mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 70 mg / mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 100 mg / mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 in the pharmaceutical composition is about 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 in the pharmaceutical composition is 150 mg / mL.

[0029] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL, 4 mg / mL, 5 mg / mL, 6 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, 10 ... 0 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL or 250 mg / mL, or any range therebetween. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds DLL3 and CD3 is about 1 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL. In some embodiments, the present invention provides an amount of the pharmaceutical composition of the present invention that is preferably at least 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, or about 250 mg / mL.

[0030] In some embodiments, the pharmaceutical composition as described in the previous item, 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., P188), polysorbate (e.g., polysorbate 20, polysorbate 80), 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, Sodium Methyl Cocoyl, Sodium Methyl Oleyl Taurate, Polyethylene Glycol, Polypropylene Glycol, Copolymer of Ethylene and Propylene Glycol, etc.

[0031] In some embodiments, as described above, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80 or poloxamer 188 (P188). In some embodiments, the surfactant is polysorbate 80.

[0032] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of polysorbate 80 is 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.1 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.2 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.3 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 1.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is 1.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.01 mg / mL, 0.05 mg / mL, 0.1 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 or 1.0 mg / mL, or any range between these points. In some embodiments, the polysorbate 80 concentration is about 0.01 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1.0 mg / mL.

[0033] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the concentration of poloxamer 188 is 0.5 mg / mL to 5 mg / mL. In some embodiments, the concentration of poloxamer 188 is 1 mg / mL to 3 mg / mL. In some embodiments, the concentration of poloxamer 188 is 1.5 mg / mL to 2.5 mg / mL. In some embodiments, the concentration of poloxamer 188 is 1.8 mg / mL to 2.2 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 2 mg / mL. In some embodiments, the concentration of poloxamer 188 is 2 mg / mL. In some embodiments, the poloxamer 188 concentration is 0.5 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.8 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5.0 mg / mL, or any range therebetween. In some embodiments, the poloxamer 188 concentration is about 0.5 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2 mg / mL, about 2.1 mg / mL, about 2.2 mg / mL, about 2.3 mg / mL, about 2.4 mg / mL, about 2.5 mg / mL, about 2.6 mg / mL, about 2.8 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, or about 5.0 mg / mL.

[0034] In some embodiments, the pharmaceutical composition as described in any of the preceding 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.

[0035] In some embodiments, the sugar is sucrose, trehalose, mannitol or sorbitol. In some embodiments, the sugar is sucrose.

[0036] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the sugar is 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of the sugar is 30 mg / mL to 100 mg / mL. In some embodiments, the concentration of the sugar is 64 mg / mL to 96 mg / mL. In some embodiments, the concentration of the sugar is 68 mg / mL to 92 mg / mL. In some embodiments, the concentration of the sugar is 72 mg / mL to 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 sugar is non-limiting and includes 10mg / mL, 20mg / mL, 30mg / mL, 35mg / mL, 37.5mg / mL, 40mg / mL, 45mg / mL, 50mg / mL, 55mg / mL, 60mg / mL, 64mg / mL, 65mg / mL, 68mg / mL, 70mg / mL, 72mg / mL, 75mg / mL, 80mg / mL, 85mg / mL, 88mg / mL, 90mg / mL, 92mg / mL, 95mg / mL, 96mg / mL, 100mg / mL or 120mg / mL, and any scope between these point values. In some embodiments, the concentration of the sugar is 30mg / mL. In some embodiments, the concentration of the sugar is 80mg / mL. In some embodiments, the concentration of the sugar is 100mg / mL. In some embodiments, the concentration of the saccharide includes, but is not limited to, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 35 mg / mL, about 37.5 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 68 mg / mL, about 70 mg / mL, about 72 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 88 mg / mL, about 90 mg / mL, about 92 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, or about 120 mg / mL.

[0037] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the sucrose is 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of the sucrose is 30 mg / mL to 100 mg / mL. In some embodiments, the concentration of the sucrose is 64 mg / mL to 96 mg / mL. In some embodiments, the concentration of the sucrose is 68 mg / mL to 92 mg / mL. In some embodiments, the concentration of the sucrose is 72 mg / mL to 88 mg / mL. In some embodiments, the concentration of the sucrose is about 30 mg / mL. In some embodiments, the concentration of the sucrose is about 80 mg / mL. In some embodiments, the concentration of the sucrose is about 100 mg / mL. In some embodiments, the concentration of described sucrose is non-limiting and is implemented to include 10mg / mL, 20mg / mL, 30mg / mL, 35mg / mL, 37.5mg / mL, 40mg / mL, 45mg / mL, 50mg / mL, 55mg / mL, 60mg / mL, 64mg / mL, 65mg / mL, 68mg / mL, 70mg / mL, 72mg / mL, 75mg / mL, 80mg / mL, 85mg / mL, 88mg / mL, 90mg / mL, 92mg / mL, 95mg / mL, 96mg / mL, 100mg / mL or 120mg / mL, and arbitrary scope between these point values. In some embodiments, the concentration of described sucrose is 30mg / mL. In some embodiments, the concentration of described sucrose is 80mg / mL. In some embodiments, the concentration of described sucrose is 100mg / mL. In some embodiments, the sucrose concentration includes, but is not limited to, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 35 mg / mL, about 37.5 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 68 mg / mL, about 70 mg / mL, about 72 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 88 mg / mL, about 90 mg / mL, about 92 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, or about 120 mg / mL.

[0038] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the buffer is 5mM to 100mM. In some embodiments, the concentration of the buffer is 10mM to 50mM. In some embodiments, the concentration of the buffer is 10mM to 30mM. In some embodiments, the concentration of the buffer is 15mM to 25mM. In some embodiments, the concentration of the buffer is 24mM to 36mM. In some embodiments, the concentration of the buffer is 27mM to 33mM. In some embodiments, the concentration of the buffer is 14mM to 22mM. In some embodiments, the concentration of the buffer is 16mM to 20mM. In some embodiments, the concentration of the buffer is about 30mM. In some embodiments, the concentration of the buffer is about 18mM. 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, 21mM, 22mM, 23mM, 24mM, 25mM, 27mM, 30mM, 33mM, 36mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, and any ranges between these point values. In some embodiments, the concentration of the buffer is 30mM. In some embodiments, the concentration of the buffer is 18mM. In some embodiments, the concentration of the buffer is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 33 mM, about 36 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0039] In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the histidine-histidine hydrochloride buffer is 5mM to 100mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 10mM to 50mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 10mM to 30mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 15mM to 25mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 24mM to 36mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 27mM to 33mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 14mM to 22mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 16mM to 20mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 18 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 27 mM, 30 mM, 33 mM, 36 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, and any ranges therebetween. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 18 mM. In some embodiments, the concentration of the histidine-histidine HCl buffer is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 33 mM, about 36 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0040] In some embodiments, the pharmaceutical composition as described in any of the preceding items further comprises an excipient; preferably, the excipient is disodium EDTA dihydrate, DTPA (diethylenetriaminepentaacetic acid), arginine hydrochloride, glycine, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, or calcium chloride; more preferably, the excipient is disodium EDTA dihydrate. In this disclosure, disodium EDTA or disodium EDTA dihydrate contains dihydrate, referred to as EDTA-2Na, with a CAS number of 6381-92-6.

[0041] In some embodiments, the pharmaceutical composition as described in any of the preceding items, 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 0.5 mg / mL; more preferably, the concentration of the excipient is 0.08 mg / mL to 0.12 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL to 0.2 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL to 0.12 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL to 0.1 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 or 1 mg / mL, and any range between these point values. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the excipient is 0.01 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the excipient is 0.1 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the excipient is 1 mg / mL. In some embodiments, the concentration of the excipient is about 0.01 mg / mL, about 0.03 mg / mL, about 0.05 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL.

[0042] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the excipient is 0.01 mg / mL to 1 mg / mL of disodium ethylenediaminetetraacetic acid dihydrate. In some embodiments, the excipient is 0.01 mg / mL to 0.5 mg / mL of disodium ethylenediaminetetraacetic acid dihydrate. In some embodiments, the excipient is 0.01 mg / mL to 0.2 mg / mL of disodium ethylenediaminetetraacetic acid dihydrate. In some embodiments, the excipient is 0.01 mg / mL to 0.12 mg / mL of disodium ethylenediaminetetraacetic acid dihydrate. In some embodiments, the excipient is 0.01 mg / mL to 0.1 mg / mL of disodium ethylenediaminetetraacetic acid dihydrate. In some embodiments, the excipient is 0.08 mg / mL to 0.12 mg / mL of disodium ethylenediaminetetraacetic acid dihydrate. In some embodiments, the excipient is 0.09 mg / mL to 0.11 mg / mL of disodium edetate dihydrate. In some embodiments, the excipient is about 0.01 mg / mL of disodium edetate dihydrate. In some embodiments, the excipient is about 0.1 mg / mL of disodium edetate dihydrate. In some embodiments, the excipient is about 1 mg / mL of disodium edetate dihydrate. In some embodiments, the concentration of the disodium edetate 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 or 1 mg / mL, and any ranges between these points. In some embodiments, the excipient is 0.01 mg / mL of disodium edetate dihydrate. In some embodiments, the excipient is 0.1 mg / mL of disodium edetate dihydrate. In some embodiments, the excipient is 1 mg / mL of disodium edetate dihydrate. In some embodiments, the concentration of disodium edetate is about 0.01 mg / mL, about 0.03 mg / mL, about 0.05 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL.

[0043] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein in the bispecific antibody that specifically binds to DLL3 and CD3:

[0044] The DLL3-VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the DLL3-VL comprises the amino acid sequence shown in SEQ ID NO: 42; and

[0045] The CD3-VH comprises the amino acid sequence of SEQ ID NO: 62, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 63.

[0046] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein in the bispecific antibody that specifically binds to DLL3 and CD3:

[0047] The amino acid sequence of the DLL3-VH is shown in SEQ ID NO: 33, and the amino acid sequence of the DLL3-VL is shown in SEQ ID NO: 42; and

[0048] The amino acid sequence of the CD3-VH is shown in SEQ ID NO: 62, and the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 63.

[0049] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the bispecific antibody specifically binds to DLL3 and CD3, wherein the Fc1 has a protrusion structure according to the knob-in-hole technique, and the Fc2 has a hole structure according to the knob-in-hole technique.

[0050] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the amino acid sequence of Fc1 in the bispecific antibody that specifically binds to DLL3 and CD3 is as shown in SEQ ID NO: 64; and the amino acid sequence of Fc2 is as shown in SEQ ID NO: 65.

[0051] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein the amino acid sequence of Fc1 in the bispecific antibody that specifically binds to DLL3 and CD3 is as shown in SEQ ID NO: 66; and the amino acid sequence of Fc2 is as shown in SEQ ID NO: 67.

[0052] In some embodiments, in the pharmaceutical composition of any of the preceding items, in which the bispecific antibody specifically binds to DLL3 and CD3, Linker 1, Linker 2, and Linker 3 are all peptide linkers known in the art, as long as the bispecific antibody exhibits the desired antigen-binding activity. For example, the peptide linker can be a flexible peptide comprising 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linker is 1-15 amino acid residues in length. In some embodiments, Linker 1 has a structure represented by the general sequence formula (GGGS)nGm, where n is 1-5, preferably 1, 2, or 3; and m is 1-10, preferably 4, 5, 6, 7, or 8. In some embodiments, the sequence of Linker 1 is GGGSGGGG (SEQ ID NO: 68). In some embodiments, Linker 2 has a structure represented by the general sequence formula Gm, where m is 1-10, preferably 1-5, and more preferably 1, 2, or 3. In some embodiments, the sequence of Linker 2 is G (SEQ ID NO: 69). In some embodiments, the linker 3 has a structure as shown in the general sequence formula (GGGGS)nGm, wherein n is 1-5, preferably 1, 2 or 3; m is 1-10, preferably 4, 5, 6, 7 or 8. In some embodiments, the sequence of linker 3 is GGGGSGGGG (SEQ ID NO: 70).

[0053] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein Linker 1, Linker 2, and Linker 3 in the bispecific antibody that specifically binds to DLL3 and CD3 are the same or different, and the amino acid sequences are each independently selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.

[0054] In some embodiments, in the pharmaceutical composition of any of the preceding items, the amino acid sequences of Linker 1, Linker 2, and Linker 3 in the bispecific antibody that specifically binds to DLL3 and CD3 are SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, respectively.

[0055] In some embodiments, the pharmaceutical composition of any of the preceding items, wherein in the bispecific antibody that specifically binds to DLL3 and CD3:

[0056] The amino acid sequence of Formula I, i.e., Chain 1, is shown in SEQ ID NO: 71, and the amino acid sequence of Formula II, i.e., Chain 2, is shown in SEQ ID NO: 72.

[0057] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0058] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0059] (b) 0.01 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188,

[0060] (c) 10 mg / mL to 120 mg / mL of sugar, and

[0061] (d) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0062] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0063] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0064] (b) 0.01 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188,

[0065] (c) 10 mg / mL to 120 mg / mL of sugar,

[0066] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0067] (e) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0068] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0069] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0070] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188,

[0071] (c) 30 mg / mL to 100 mg / mL of sucrose, and

[0072] (d) 10 mM to 50 mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, or citric acid-sodium citrate buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0073] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0074] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0075] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188,

[0076] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0077] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0078] (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer, or citric acid-sodium citrate buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0079] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0080] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0081] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or 1 mg / mL to 3 mg / mL of poloxamer 188,

[0082] (c) 30 mg / mL to 100 mg / mL of sucrose, and

[0083] (d) 10 mM to 50 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0084] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0085] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0086] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or 1 mg / mL to 3 mg / mL of poloxamer 188,

[0087] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0088] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0089] (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0090] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0091] (a) 70 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0092] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80 or 1 mg / mL to 3 mg / mL of poloxamer 188,

[0093] (c) 64 mg / mL to 96 mg / mL of sucrose, and

[0094] (d) 10 mM to 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0095] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0096] (a) 70 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0097] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80 or 1 mg / mL to 3 mg / mL of poloxamer 188,

[0098] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0099] (d) 0.01 mg / mL to 0.5 mg / mL of disodium edetate dihydrate, and

[0100] (e) 10 mM to 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0101] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0102] (a) 80 mg / mL to 120 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0103] (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80 or 1.5 mg / mL to 2.5 mg / mL of poloxamer 188,

[0104] (c) 64 mg / mL to 96 mg / mL of sucrose, and

[0105] (d) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0106] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0107] (a) 80 mg / mL to 120 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0108] (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80 or 1.5 mg / mL to 2.5 mg / mL of poloxamer 188,

[0109] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0110] (d) 0.08 mg / mL to 0.12 mg / mL of disodium edetate dihydrate, and

[0111] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0112] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0113] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0114] (b) 0.01 mg / mL to 1.0 mg / mL of polysorbate 80,

[0115] (c) 10 mg / mL to 120 mg / mL of sugar,

[0116] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0117] (e) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0118] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0119] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0120] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80,

[0121] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0122] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0123] (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0124] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0125] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0126] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80,

[0127] (c) 30 mg / mL to 100 mg / mL of sucrose, and

[0128] (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0129] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0130] (a) 70 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0131] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80,

[0132] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0133] (d) 0.01 mg / mL to 0.5 mg / mL of disodium edetate dihydrate, and

[0134] (e) 10 mM to 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0135] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0136] (a) 80 mg / mL to 120 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0137] (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80,

[0138] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0139] (d) 0.08 mg / mL to 0.12 mg / mL of disodium edetate dihydrate, and

[0140] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0141] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0142] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0143] (b) 0.5 mg / mL to 5 mg / mL Poloxamer 188,

[0144] (c) 10 mg / mL to 120 mg / mL of sugar,

[0145] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0146] (e) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0147] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0148] (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0149] (b) 1 mg / mL to 3 mg / mL Poloxamer 188,

[0150] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0151] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0152] (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0153] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0154] (a) 70 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0155] (b) 1 mg / mL to 3 mg / mL Poloxamer 188,

[0156] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0157] (d) 0.01 mg / mL to 0.5 mg / mL of disodium edetate dihydrate, and

[0158] (e) 10 mM to 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0159] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0160] (a) 80 mg / mL to 120 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0161] (b) 1.5 mg / mL to 2.5 mg / mL Poloxamer 188,

[0162] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0163] (d) 0.08 mg / mL to 0.12 mg / mL of disodium edetate dihydrate, and

[0164] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0165] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0166] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0167] (b) about 0.4 mg / mL of polysorbate 80,

[0168] (c) about 80 mg / mL of sucrose,

[0169] (d) about 0.1 mg / mL of disodium edetate dihydrate, and

[0170] (e) about 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0171] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0172] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0173] (b) about 0.4 mg / mL of polysorbate 80,

[0174] (c) about 80 mg / mL of sucrose,

[0175] (d) about 0.1 mg / mL of disodium edetate dihydrate, and

[0176] (e) about 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0177] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0178] (a) 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0179] (b) 0.4 mg / mL of polysorbate 80,

[0180] (c) 80 mg / mL sucrose,

[0181] (d) 0.1 mg / mL of disodium edetate dihydrate, and

[0182] (e) 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0183] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0184] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0185] (b) about 0.4 mg / mL of polysorbate 80,

[0186] (c) about 80 mg / mL of sucrose,

[0187] (d) about 0.1 mg / mL of disodium edetate dihydrate, and

[0188] (e) about 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0189] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0190] (a) 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0191] (b) 0.4 mg / mL of polysorbate 80,

[0192] (c) 80 mg / mL sucrose,

[0193] (d) 0.1 mg / mL of disodium edetate dihydrate, and

[0194] (e) 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0195] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0196] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0197] (b) about 0.4 mg / mL of polysorbate 80,

[0198] (c) about 80 mg / mL of sucrose,

[0199] (d) about 0.1 mg / mL of disodium edetate dihydrate, and

[0200] (e) about 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0201] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0202] (a) 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0203] (b) 0.4 mg / mL of polysorbate 80,

[0204] (c) 80 mg / mL sucrose,

[0205] (d) 0.1 mg / mL of disodium edetate dihydrate, and

[0206] (e) 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0207] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0208] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0209] (b) about 0.4 mg / mL of polysorbate 80,

[0210] (c) about 80 mg / mL of sucrose, and

[0211] (d) about 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0212] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0213] (a) 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0214] (b) 0.4 mg / mL of polysorbate 80,

[0215] (c) 80 mg / mL sucrose, and

[0216] (d) 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0217] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0218] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0219] (b) about 0.4 mg / mL of polysorbate 80,

[0220] (c) about 80 mg / mL of sucrose, and

[0221] (d) about 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0222] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0223] (a) 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0224] (b) 0.4 mg / mL of polysorbate 80,

[0225] (c) 80 mg / mL sucrose, and

[0226] (d) 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0227] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0228] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0229] (b) about 0.4 mg / mL of polysorbate 80,

[0230] (c) about 80 mg / mL of sucrose, and

[0231] (d) about 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0232] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0233] (a) 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0234] (b) 0.4 mg / mL of polysorbate 80,

[0235] (c) 80 mg / mL sucrose, and

[0236] (d) 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0237] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0238] (a) about 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0239] (b) about 2 mg / mL of Poloxamer 188,

[0240] (c) about 80 mg / mL of sucrose,

[0241] (d) about 0.1 mg / mL of disodium edetate dihydrate, and

[0242] (e) about 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0243] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0244] (a) 100 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0245] (b) 2 mg / mL of Poloxamer 188,

[0246] (c) 80 mg / mL sucrose,

[0247] (d) 0.1 mg / mL of disodium edetate dihydrate, and

[0248] (e) 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0249] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0250] (a) about 70 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0251] (b) about 2 mg / mL of Poloxamer 188,

[0252] (c) about 80 mg / mL of sucrose,

[0253] (d) about 0.1 mg / mL of disodium edetate dihydrate, and

[0254] (e) about 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.5.

[0255] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0256] (a) 70 mg / mL of a bispecific antibody that specifically binds to DLL3 and CD3,

[0257] (b) 2 mg / mL of Poloxamer 188,

[0258] (c) 80 mg / mL sucrose,

[0259] (d) 0.1 mg / mL of disodium edetate dihydrate, and

[0260] (e) 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.5.

[0261] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0262] (a) 1 mg / mL to 20 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0263] (b) 0.01 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188,

[0264] (c) 10 mg / mL to 120 mg / mL of sugar,

[0265] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0266] (e) 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0;

[0267] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0268] (a) 1 mg / mL to 10 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0269] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188,

[0270] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0271] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0272] (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer or citric acid-sodium citrate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 5.5.

[0273] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0274] (a) 1 mg / mL to 10 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0275] (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80,

[0276] (c) 30 mg / mL to 100 mg / mL of sucrose,

[0277] (d) 0.01 mg / mL to 1 mg / mL of disodium edetate dihydrate, and

[0278] (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 4.5 to 5.5.

[0279] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0280] (a) 4 mg / mL to 6 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0281] (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80 or 1 mg / mL to 3 mg / mL of poloxamer 188,

[0282] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0283] (d) 0.01 mg / mL to 0.5 mg / mL of disodium edetate dihydrate, and

[0284] (e) 10 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 4.5 to 5.5.

[0285] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0286] (a) 4 mg / mL to 6 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0287] (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80,

[0288] (c) 64 mg / mL to 96 mg / mL of sucrose,

[0289] (d) 0.08 mg / mL to 0.12 mg / mL of disodium edetate dihydrate, and

[0290] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 4.8 to 5.2.

[0291] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0292] (a) about 5 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0293] (b) about 0.4 mg / mL of polysorbate 80,

[0294] (c) about 80 mg / mL of sucrose,

[0295] (d) about 0.1 mg / mL of disodium edetate dihydrate, and

[0296] (e) about 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0297] In some embodiments, the pharmaceutical composition as described in any of the preceding items comprises the following components:

[0298] (a) 5 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3,

[0299] (b) 0.4 mg / mL of polysorbate 80,

[0300] (c) 80 mg / mL sucrose,

[0301] (d) 0.1 mg / mL of disodium edetate dihydrate, and

[0302] (e) 18 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0303] 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.

[0304] In one aspect, the present disclosure provides a method for preparing a lyophilized formulation, comprising the step of freeze-drying the pharmaceutical composition as described in any one of the preceding items.

[0305] In one aspect, the present disclosure provides a lyophilized formulation obtained by the method as described above.

[0306] In one aspect, the present disclosure provides a pharmaceutical composition as described in any of the preceding items, which is a subcutaneous injection formulation, an intravenous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution as described in any of the preceding items is a subcutaneous injection formulation.

[0307] In some embodiments, the pharmaceutical composition, lyophilized formulation or reconstituted solution as described in any of the preceding items is suitable for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation or reconstituted solution as described in any of the preceding items is suitable for subcutaneous injection.

[0308] In some embodiments, the pharmaceutical composition, lyophilized formulation or reconstituted solution as described in any of the preceding items is used to prepare a drug for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation or reconstituted solution as described in any of the preceding items is used to prepare a drug for subcutaneous injection.

[0309] The present disclosure also provides a pharmaceutical composition as described in any of the preceding items, or a lyophilized formulation as described in any of the preceding items, or an injectable formulation as described in any of the preceding items for use as a medicament. In some embodiments, the medicament is used to treat a tumor or cancer.

[0310] The present disclosure also provides a method for treating tumors or cancer, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition as described in any of the preceding items, or the lyophilized formulation as described in any of the preceding items, or the injectable formulation as described in any of the preceding items.

[0311] In one aspect, the present disclosure provides use of the composition as described in any of the preceding items, or the lyophilized formulation as described in any of the preceding items, or the injectable formulation as described in any of the preceding items in the preparation of a medicament for treating tumors or cancer.

[0312] In some embodiments, the tumor or cancer described in any of the preceding items is selected from the group consisting of lung cancer, small cell lung cancer, large cell lung cancer, head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, large cell lung cancer, triple-negative breast cancer, and lymphoma.

[0313] In some embodiments, the tumor or cancer as described in any of the preceding items is a solid tumor.

[0314] In some embodiments, the tumor or cancer as described in any of the preceding items is lung cancer.

[0315] In some embodiments, the tumor or cancer as described in any of the preceding items is small cell lung cancer.

[0316] In some embodiments, the method for treating a tumor or cancer as described in any of the preceding items further comprises the use of a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an anti-tumor agent, radiotherapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an anti-tumor agent, an immune checkpoint inhibitor, or a combination thereof.

[0317] In some embodiments, the method for treating tumors or cancer as described above, wherein the second therapeutic agent is administered simultaneously, sequentially, or separately with the bispecific antibody formulation described in any one of the present disclosures.

[0318] The bispecific antibody preparations provided herein have the characteristics of good therapeutic activity, safety, pharmacokinetic properties and drugability (such as stability). BRIEF DESCRIPTION OF THE DRAWINGS

[0319] FIG1 shows a schematic diagram of the structure of Format 3 of the DLL3-CD3 bispecific antibody.

[0320] Figure 2A shows the experimental results of the binding of bispecific antibodies to DLL1. The results show that the DLL3-CD3 bispecific antibodies disclosed herein do not bind to DLL1.

[0321] Figure 2B shows the experimental results of the binding of bispecific antibodies to DLL4. The results show that the DLL3-CD3 bispecific antibodies disclosed herein do not bind to DLL4.

[0322] Figure 3 shows the results of the cell-killing experiment of the bispecific antibody. The results show that the bispecific antibody disclosed herein has no killing effect on H460 cells that do not express DLL3.

[0323] FIG4A shows the experimental results of the bispecific antibody activation of T cells, which show that the bispecific antibody disclosed herein has a significant effect of activating T cells in the presence of DLL3 / H82 cells expressing DLL3.

[0324] FIG4B shows that the bispecific antibodies of the present disclosure do not activate T cells in the presence of negative cells H460 that do not express DLL3.

[0325] FIG5A shows the experimental results of cytokine release of the bispecific antibody, which shows that in the presence of H1184 cells, the bispecific antibody of the present disclosure stimulates PBMC cells to secrete very low levels of IFNγ.

[0326] FIG5B shows that in the presence of H1184 cells, the bispecific antibodies of the present disclosure stimulated PBMC cells to secrete very low levels of IL-6. DETAILED DESCRIPTION

[0327] the term

[0328] The terms used herein are for the purpose of describing the embodiments only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0329] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including, but not limited to." Unless otherwise stated, "comprising" includes "consisting of."

[0330] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0331] The term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both meanings or either meaning.

[0332] Unless designated as being from a non-human species (e.g., "mouse DLL3," "mouse DLL3 fragment," "monkey DLL3," "monkey DLL3 fragment," etc.), "DLL3" and "DLL3 fragment" as used herein refer to the well-known human DLL3 protein or a fragment thereof.

[0333] Unless specified as being from a non-human species (e.g., "mouse CD3," "mouse CD3 fragment," "monkey CD3," "monkey CD3 fragment," etc.), "CD3" and "CD3 fragment" as used herein refer to the well-known human CD3 protein or a fragment thereof.

[0334] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0335] The term "amino acid mutation" includes amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to achieve the final construct, as long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, are also useful. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has mutated from T to W.

[0336] The term "antibody" is used in the broadest sense and encompasses 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 antibody fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Depending on the context, a skilled person can determine the specific meaning of "antibody".

[0337] "Natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light chains and two heavy chains bound by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH, also known as variable heavy domain, heavy chain variable region), followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL, also known as variable light domain, or light chain variable domain), followed by a constant light domain (light chain constant region, CL). The terms "full-length antibody", "complete antibody" and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of natural antibody structure or having a heavy chain containing an Fc region as defined herein.

[0338] 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 at least two different antigenic epitopes of the same antigen. Prior art has disclosed bispecific antibodies of various structures. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies; based on the number of antigen-binding regions, they can be divided into bivalent, trivalent, tetravalent, or higher-valent bispecific antibodies; and based on whether the structure is bilaterally symmetrical, they can be divided into symmetrical bispecific antibodies and asymmetrical bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, are formed by combining two or more Fab fragments into one molecule. They have 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 and other bispecific antibodies; IgG-like bispecific antibodies (for example, with Fc fragments) have a relatively large molecular weight. The Fc fragment helps in the later purification of the antibody and improves its solubility and stability. The Fc part may also bind to the receptor FcRn to increase 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).

[0339] 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.

[0340] Table 1. Relationships between CDR numbering systems

[0341] Unless otherwise specified, the variable region and CDR sequences in the disclosed embodiments are all numbered using the "Kabat" convention.

[0342] The term "antibody fragment" or "antigen-binding 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.

[0343] 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.

[0344] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0345] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., the constant region and the framework region portion of the variable region).

[0346] The term "affinity" refers to the overall intensity of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its part Y can generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art (including those described herein). The term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" as used herein is intended to refer to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "KD" refers to an equilibrium dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and is expressed as molar concentration (M). The KD value of an antibody can be measured using methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).

[0347] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for possible natural mutations that may be present in small amounts. In contrast, polyclonal antibody preparations typically contain a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes. In some embodiments, the antibodies provided herein are monoclonal antibodies.

[0348] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent of an antigen binding molecule (e.g., an antibody). An antigen may have one or more epitopes that can interact with different antigen binding molecules (e.g., antibodies).

[0349] The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody or its antigen-binding fragment. An epitope can be formed by contiguous amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, when the non-contiguous amino acids are brought into spatial proximity due to the folding of the antigen (i.e., by the tertiary folding of the antigen in the case of proteinaceous properties). Conformational epitopes differ from linear epitopes in that antibody binding to the conformational epitope is lost in the presence of denaturing solvents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0350] The terms "capable of specific binding", "specific binding" or "binding" refer to the ability of an antigen-binding molecule 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 4 M or less. In some embodiments, the antibody binds to the antigen with a KD that is 10% or less (e.g., 1%) of the KD of the antibody bound to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as those measured by FACS or surface plasmon resonance assays. However, an antibody that specifically binds to an antigen or its epitope may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous) such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp), or Callithrix jacchus (common marmoset, marmoset).

[0351] The term "not binding" means that the antigen-binding molecule is unable to bind to an antigen or its epitope in the manner of specific binding described above. For example, when the antibody is about 1×10 -6The present invention binds to the antigen or its epitope with an equilibrium dissociation constant (KD) of M or greater.

[0352] The term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to a target antigen. Specific antigen binding moieties include the antigen binding domains of antibodies, for example, comprising heavy chain variable regions and light chain variable regions. The term "antigen binding moiety that specifically binds to CD3" refers to a module that is capable of binding to CD3 or its epitope with sufficient affinity so that the molecule containing the module can be used as a diagnostic and / or therapeutic agent targeting CD3. For example, an antigen binding moiety that specifically binds to CD3 has the following equilibrium dissociation constant (KD): <about 10 nM, which is measured by surface plasmon resonance assay. Antigen binding moieties include antibody fragments as defined herein, such as Fab, substituted Fab or scFv.

[0353] The term "linker" refers to a connecting unit that connects two polypeptide fragments. As used herein, linkers appearing in the same structural formula may be the same or different. A linker may be a peptide linker comprising one or more amino acids, typically comprising about 1-30, 2-24, or 3-15 amino acids. The linkers used herein may be the same or different. When "-" appears in a structural formula, it indicates that the units on either side are directly connected by a covalent bond.

[0354] "Tm" is the melting denaturation temperature (intrinsic fluorescence). When a protein denatures (by heat or denaturants), the tertiary structure opens up, the microenvironment of the aromatic amino acids changes, and the emitted fluorescence spectrum shifts. For purposes of this disclosure, Tm1 refers to the temperature at which fluorescence reaches half its maximum value.

[0355] "Tonset" is the denaturation onset temperature. It refers to the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.

[0356] "Tagg" stands for aggregation onset temperature. Aggregates are detected by static light scattering at two wavelengths, 266 nm and 473 nm, to measure the temperature at which the sample begins to aggregate. Tagg 266 refers to the aggregation onset temperature measured at 266 nm.

[0357] It should be understood that although no specific coding nucleotide sequence is provided in the present disclosure, a skilled person can determine the corresponding coding nucleotide sequence based on the amino acid sequence according to codon rules and host codon preferences.

[0358] The term "fused" or "linked" means that the components (eg, an antigen binding moiety and an Fc domain) are covalently linked directly or via a linker.

[0359] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell in which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence for guiding and / or controlling (together with the host cell) the expression of one or more heterologous coding regions operably connected thereto. An expression construct can include but is not limited to affecting or controlling transcription, translation and affecting the sequence of RNA splicing of the coding region operably connected thereto when introns are present.

[0360] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0361] The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless explicitly stated, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cynomolgus monkey" or "cynomolgus monkey" refers to cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

[0362] "Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0363] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.

[0364] "Treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions attempted to be applied to the individual being treated, and can be performed for preventive purposes or during the course of clinical pathology. 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, ameliorating or palliating the disease state, and regression or improved prognosis.

[0365] The terms "recurrence," "relapse," and "relapsed" refer to the return of a cancer or disease after clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence may be considered a relapse.

[0366] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent the occurrence of symptoms and / or their potential causes, and / or ameliorate or improve the damage caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0367] A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptoms, particularly a state or symptoms associated with the disease state, or otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable symptoms in any way associated with the disease.

[0368] A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or preventive effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations. "Therapeutically effective amount" and "prophylactically effective amount" may vary depending on a variety of factors: such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the subject.

[0369] "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.

[0370] "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.

[0371] "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.

[0372] 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.

[0373] "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.

[0374] 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.

[0375] "Poloxamer" is an α-hydrogen-ω-hydroxy poly(ethylene oxide)a-poly(propylene oxide)b-poly(ethylene oxide)a block copolymer. Propylene oxide and propylene glycol react to form polyoxypropylene glycol, and then ethylene oxide is added to form the block copolymer. Here, a is the number of ethylene oxide units, and b is the number of propylene oxide units. Examples of poloxamers include, but are not limited to, poloxamer 188 (P188 or PF68). Specifically, poloxamer 188 has 75 to 85 ethylene oxide units (a), 25 to 30 propylene oxide units (b), an ethylene oxide (EO) content of 79.9% to 83.7%, and an average molecular weight of 7680 to 9510.

[0376] "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.

[0377] 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.

[0378] 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.

[0379] 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).

[0380] 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.).

[0381] 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.

[0382] Substitution, insertion, and deletion variants

[0383] In certain embodiments, antigen binding molecule variants with one or more amino acid substitutions are provided. Substitutions can be made in CDR and FR. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 2. More substantial variations are provided under the heading of "exemplary substitutions" in Table 2, and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the desired activity of the product screening is determined, such as the antigen binding to which retention / improvement is sought, the immunogenicity to which reduction is sought, or the ADCC or CDC to which improvement is sought.

[0384] Table 2. Amino acid substitutions

[0385] Based on common side chain properties, amino acids can be grouped as follows:

[0386] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0387] (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0388] (3) Acidic: Asp, Glu;

[0389] (4) Basic: His, Lys, Arg;

[0390] (5) Residues that affect chain orientation: Gly, Pro;

[0391] (6) Aromatic: Trp, Tyr, Phe.

[0392] Non-conservative substitutions would involve exchanging a member from one class for a member from another class.

[0393] A class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., humanized or human antibody). Generally, the variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be readily produced using, for example, affinity maturation techniques based on phage display (e.g., those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on phage, and screened for specific biological activity (e.g., binding affinity). Changes (e.g., substitutions) can be made to CDR, for example, to improve antibody affinity. Such changes can be made to CDR "hot spots," i.e., residues encoded by codons that undergo mutations at high frequency during the somatic maturation process, and / or residues contacting antigens, while binding affinity is tested for the variant VH or VL obtained. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., fallibility PCR, chain shuffling, or oligonucleotide-guided mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variant with desired affinity. Another method of introducing diversity relates to a CDR-directed method in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues that specifically identify antigen binding can be, for example, identified using alanine scanning mutagenesis or modeling.

[0394] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to the CDRs that do not substantially reduce binding affinity. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

[0395] A method that can be used to identify residues or regions that can be used as mutagenesis targets in antibodies is called "alanine scanning mutagenesis". In this method, a residue or residue group (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., Ala or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. In addition, the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain desired properties.

[0396] Amino acid sequence insertions include: fusions of one residue or polypeptides of 100 or more residues in length to the amino and / or carboxyl termini; and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of enzymes (or polypeptides that extend the serum half-life of the antibody) to the N- or C-termini of the antibody.

[0397] Renovation of the Fc region

[0398] In one aspect, the Fc region of the bispecific antibody of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, such as its binding to an Fcγ receptor, and reduce or eliminate effector function. A native IgG Fc region, specifically an IgG1 Fc region or an IgG4 Fc region, may cause the bispecific antibody of the present disclosure to target cells expressing Fc receptors rather than cells expressing antigens. The modified Fc region of the present disclosure exhibits reduced binding affinity to Fc receptors and / or reduced effector function. In some embodiments, the modified Fc region has a binding affinity to Fc receptors that is decreased by 50%, 80%, 90% or more than 95% compared to the native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, or FcγRIIIa. In some embodiments, the Fc region of the transformation has a reduced binding affinity for complement, such as C1q, compared to the native Fc region. In some embodiments, the Fc region of the transformation has a reduced binding affinity for neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the Fc region of the transformation has reduced effector functions, which can include but are not limited to one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming. For an IgG1 Fc region, substitutions of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329, among others, can reduce effector function. In some embodiments, the Fc region is a human IgG1 Fc region, and amino acid residues at positions 234 and 235 are A, numbering is according to the EU index. For an IgG4 Fc region, substitutions of amino acid residues at positions 228, among others, can reduce effector function.

[0399] When the bispecific antibody comprises different binding modules fused to the two subunits in the Fc region, undesirable homodimerization may result. In order to improve yield and purity, it is therefore advantageous to introduce modifications that promote heterodimerization in the Fc region of the bispecific antibody of the present disclosure. In some embodiments, the Fc region of the present disclosure comprises the transformation according to knob-into-hole (KIH) technology, which involves introducing a protrusion structure (knob) at the interface of the first subunit and introducing a hole structure (hole) at the interface of the second subunit. The protrusion structure is positioned in the hole structure, promoting the formation of heterodimers and inhibiting the production of homodimers. The protrusion structure is constructed by replacing the small amino acid side chains from the interface of the first subunit with larger side chains (such as tyrosine or tryptophan). The hole structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine). The protrusion structure and the hole structure are prepared by changing the nucleic acid encoding the polypeptide, and optional amino acid substitutions are shown in the following table:

[0400] Table 3. KIH mutation combinations

[0401] In addition to the knob-in-hole technique, other techniques for modifying the CH3 domain of the heavy chain to achieve heterodimerization are also known in the art, such as WO1996027011A1, WO1998050431, EP1870459, WO2007110205, WO2009089004, WO2010129304, WO201190754, WO2011143545, WO2012058768, WO2013157954 and WO2013096291.

[0402] The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues are removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Therefore, in some embodiments, a composition of intact antibodies can include a population of antibodies in which all K447 residues and / or G446+K447 residues have been removed. In some embodiments, a composition of intact antibodies can include a population of antibodies in which the K447 residue and / or G446+K447 residues have not been removed. In some embodiments, a composition of intact antibodies has a population of antibodies that are a mixture of antibodies with and without the K447 residue and / or G446+K447 residues.

[0403] Recombination methods

[0404] Bispecific antibodies can be produced using recombinant methods.For these methods, one or more isolated nucleic acids encoding the bispecific antibodies are provided.

[0405] In the case of a natural antibody, a natural antibody fragment or a bispecific antibody with a homodimer heavy chain, two nucleic acids are required, one for a light chain or its fragment and one for a heavy chain or its fragment. Such nucleic acid encoding comprises the amino acid sequence of antibody VL and / or comprises the amino acid sequence of antibody VH (e.g., light chain and / or heavy chain of an antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0406] In the case of a bispecific antibody with a heterodimeric heavy chain, for example, four nucleic acids are required: one for the first light chain, one for the first heavy chain comprising a first heterologous monomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors, typically these nucleic acids are located on two or three expression vectors, i.e., one vector can contain more than one of these nucleic acids.

[0407] In one embodiment, present disclosure provides the nucleic acid of the separation of encoding antibody as described above.Such nucleic acid can encode any of the aforementioned polypeptide chains independently.On the other hand, present disclosure provides one or more vectors (such as expression vectors) comprising such nucleic acid.On the other hand, present disclosure provides the host cell comprising such nucleic acid.In one embodiment, there is provided a method for preparing bispecific antibodies, wherein said method comprises, under conditions suitable for antibody expression, cultivating a host cell comprising the nucleic acid encoding the antibody, as provided above, and optionally reclaiming the antibody from host cell (or host cell culture medium).

[0408] In order to recombinantly produce bispecific antibodies, the nucleic acid encoding the protein is separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody), or produced by recombinant methods or obtained by chemical synthesis.

[0409] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required for the antibodies. Following expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0410] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. Suitable host cells for expressing (glycosylated) antibodies can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, for example, US5959177, US6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted for growth in suspension. Other examples of suitable mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0411] Examples and test cases

[0412] The present disclosure is further described below with reference to the following examples and test cases, but these examples and test cases are not intended to limit the scope of this disclosure. Experimental methods in the examples and test cases disclosed herein, where specific conditions are not specified, generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents whose sources are not specified are commercially available reagents.

[0413] 1. Preparation of bispecific antibodies that specifically bind to DLL3 and CD3

[0414] Example 1. Preparation of DLL3 antigen, detection protein, and stably transfected cell lines

[0415] 1.1 Construction of cell lines with high expression of DLL3

[0416] The pCDH lentiviral expression vector plasmid (synthesized by GENEWIZ) containing SEQ ID NOs: 1-2 was transfected with the pVSVG and pCMV lentiviral packaging vectors, respectively, into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) using Lipofectamine 3000 (Invitrogen, L3000015). The virus-containing supernatant was collected, filtered, and subjected to ultracentrifugation. The supernatant was discarded and the cells were resuspended in 0.2 mL of sterile PBS. The concentrated virus was used to infect Chinese hamster ovary (CHO-S) cells (Invitrogen, R80007), DMS53 (ATCC, CRL-2062), and H82 (ATCC, HTB-175). After selection with puromycin for two to three weeks, cells were isolated by FACS. Selected monoclonal cell lines were expanded and cryopreserved for subsequent experiments.

[0417] The pCDH-CMV-MCS-EF1-puro lentiviral expression vector plasmid (synthesized by GENEWIZ) containing the LUC and GFP genes was used to transfect 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) with the pCDH plasmid and pVSVG and pCMV lentiviral packaging vectors, respectively, using Lipofectamine 3000 (Invitrogen, L3000015). The supernatant containing the virus was collected, filtered, and ultracentrifuged. The supernatant was discarded and resuspended in 0.2 mL of sterile PBS. The concentrated virus was used to infect SHP77 (ATCC, CRL-2195), H1184 (ATCC, CRL-5858), and H460 (Chinese Academy of Sciences Cell Bank, TCHu205). After puromycin selection for two to three weeks, single-cell FACS sorting was performed. Selected monoclonal cell lines were expanded and cryopreserved for subsequent experiments.

[0418] The relevant protein sequences used are as follows:

[0419] 1. Human DLL3 full-length protein (SEQ ID NO: 1):

[0420] 2. Cynomolgus monkey DLL3 full-length protein (SEQ ID NO: 2):

[0421] 3. Rat DLL3 full-length protein (SEQ ID NO: 3):

[0422] 4. Mouse DLL3 full-length protein (SEQ ID NO: 4):

[0423] 5. Human DLL1 full-length protein (SEQ ID NO: 5):

[0424] 6. Human DLL4 full-length protein (SEQ ID NO: 6):

[0425] 1.2 Preparation of Antigens

[0426] Using human DLL3 (Uniprot, Q9NYJ7), cynomolgus macaque DLL3 (Uniprot, A0A2K5WSR4), and mouse DLL3 (Uniprot, O88516) sequences as templates, human DLL3 ECD fusion proteins containing different tags were designed and cloned into the pTT5 vector. After expression in 293E cells, the antigens were obtained. The amino acid sequences of the relevant proteins are as follows:

[0427] 1.His-hDLL3(ECD)(SEQ ID NO:7):

[0428] Note: The dot-dashed line represents the signal peptide sequence, the single dash line represents the his tag and linker, and the double dash line represents the DLL3 extracellular region.

[0429] 2.Fc-hDLL3(ECD)(SEQ ID NO:8):

[0430] Note: The dot-dashed line represents the signal peptide sequence, the single dash line represents the Fc tag and linker, and the double dash line represents the DLL3 extracellular region.

[0431] 3.hDLL3(ECD)-strep twin(SEQ ID NO:9):

[0432] Note: The dot-dashed line represents the signal peptide sequence, the double-dashed line represents the DLL3 extracellular region, and the single-dashed line represents the strep twin tag.

[0433] 4.cynoDLL3(ECD)-strep twin(SEQ ID NO:10):

[0434] Note: The dot-dashed line represents the signal peptide sequence, the double-dashed line represents the DLL3 extracellular region, and the single-dashed line represents the strep twin tag.

[0435] 5.mouDLL3(ECD)-strep twin(SEQ ID NO:11):

[0436] Note: The dot-dashed line represents the signal peptide sequence, the double-dashed line represents the DLL3 extracellular region, and the single-dashed line represents the strep twin tag.

[0437] Example 2. Preparation of anti-human DLL3 antibodies

[0438] Monoclonal antibodies against human DLL3 were prepared using hybridoma technology. The immunization method was as follows:

[0439] The first group used His-hDLL3 (ECD) (SEQ ID NO: 7) as the immunogen. Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No.77161) adjuvant cross immunization. Antigen and adjuvant ( Gold Adjuvant) ratio was 1:1, antigen and adjuvant (Thermo The ratio of immunization dose to Alum was 3:1, 50 μg / mouse / time (primary immunization) and 25 μg / mouse / time (boosting immunization). The antibody titer in the mouse serum was determined by ELISA.

[0440] The second group of mice were immunized with DLL3-CHO-s cells and Fc-hDLL3 (ECD) (SEQ ID NO: 8) by alternating immunization with cell-based and protein-based antigens. For cell-based immunization, each mouse was intraperitoneally injected with 0.1 mL of saline diluted to 10 μg / mL. 8 / mL concentration of cell fluid. Fc-hDLL3 (ECD) antigen was used Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No.77161) adjuvant cross immunization. Antigen and adjuvant ( Gold Adjuvant) ratio was 1:1, antigen and adjuvant (Thermo The ratio of immunization dose to Alum was 3:1, 50 μg / mouse / time (primary immunization) and 25 μg / mouse / time (boosting immunization). The antibody titer in the mouse serum was determined by ELISA.

[0441] The optimized electrofusion method was used to combine spleen lymphocytes with myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) were fused to obtain hybridoma cells. Based on the hybridoma cell growth density, hybridoma culture supernatants were assayed using ELISA for binding to DLL3 protein and FACS for binding to DLL3 CHO-S cells. Clones that bound to human DLL3 protein, monkey DLL3 protein, and DLL3 CHO-S cells, while not binding to wild-type CHO-S cells, were selected for cryopreservation, expansion, and one or two subclones until single-cell clones were obtained. Through these experimental screening procedures, hybridoma clone mAb6 was obtained.

[0442] The hybridoma clones were expanded and cultured, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using degenerate primers of mouse-Ig to finally obtain the variable region sequence of the antibody.

[0443] mAb6 Heavy Chain Variable Region (SEQ ID NO: 12):

[0444] mAb6 light chain variable region (SEQ ID NO: 13):

[0445] Table 4. Antibody CDRs

[0446] Note: The amino acid residues of the VH / VL CDRs are determined and annotated according to the Kabat numbering system.

[0447] The heavy chain variable region and light chain variable region of the mouse antibody were cloned into the pTT5 vector plasmid containing the human IgG1 heavy chain constant region shown in SEQ ID NO: 20 and the kappa light chain constant region shown in SEQ ID NO: 21, respectively, and then transfected into HEK293 cells to obtain the anti-DLL3 chimeric antibody M6CHI.

[0448] Human IgG1 heavy chain constant region (SEQ ID NO: 20):

[0449] Human kappa light chain constant region (hκ) (SEQ ID NO: 21):

[0450] Example 3. Humanization of anti-DLL3 monoclonal antibodies

[0451] By comparing the Kabat human antibody heavy and light chain variable region germline gene database, the heavy and light chain variable region germline genes with high homology were selected as templates, and the CDRs of the mouse antibody were grafted into the corresponding human templates to form a variable region sequence with the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acid substitutions were made to certain amino acids in the variable region, and then the variable region was recombined with the constant region (exemplarily, with the human IgG1 heavy chain constant region shown in SEQ ID NO: 20 and the human kappa light chain constant region shown in SEQ ID NO: 21) to obtain a full-length humanized antibody.

[0452] The human germline light chain template of mAb6 antibody is IGKV1-16*01 or IGKV3-20*02 and IGKJ4*01, and the human germline heavy chain template is IGHV1-3*01, IGHV7-4-1*02 or IGHV3-73*01 and IGHJ6*01. Optionally, amino acid residues at positions 1, 36, 42, 43, 44, 46, 52, 56, 69, 71, 79, 85, and / or 94 of the light chain variable region of the humanized antibody are substituted; and / or amino acid residues at positions 1, 27, 30, 38, 43, 48, 67, 68, 69, 71, 73, 75, 76, 93, 56, 58, and / or 5 of the heavy chain variable region of the humanized antibody are substituted.

[0453] Table 5. Humanized templates and corresponding point mutations of mAb6 mouse antibody

[0454] Note: The amino acid positions in the table are numbered according to Kabat. F36L means that the F at position 36 is mutated to L according to the Kabat numbering system. The same applies below.

[0455] The obtained sequence of the hu6 humanized antibody variable region is as follows:

[0456] hu6 VH1 (SEQ ID NO: 22)

[0457] hu6 VH2 (SEQ ID NO: 23)

[0458] hu6 VH3 (SEQ ID NO: 24)

[0459] hu6 VH4 (SEQ ID NO: 25)

[0460] hu6 VH5(SEQ ID NO:26)

[0461] hu6 VH6(SEQ ID NO:27)

[0462] hu6 VH7(SEQ ID NO:28)

[0463] hu6VH8(SEQ ID NO:29)(Graft IGHV1-3*01)

[0464] hu6VH9(SEQ ID NO:30)(Graft IGHV7-4-1*02)

[0465] hu6VH10(SEQ ID NO:31)(Graft IGHV3-73*01)

[0466] hu6VH11(SEQ ID NO:32)

[0467] hu6VH12(SEQ ID NO:33)

[0468] hu6VH13(SEQ ID NO:34)

[0469] hu6VH14(SEQ ID NO:35)

[0470] hu6VH15(SEQ ID NO:36)

[0471] hu6 VL1(SEQ ID NO:37)

[0472] hu6 VL2(SEQ ID NO:38)

[0473] hu6 VL3(SEQ ID NO:39)

[0474] hu6 VL4(SEQ ID NO:40)

[0475] hu6VL5(SEQ ID NO:41)(Graft IGKV1-16*01)

[0476] hu6VL6 (SEQ ID NO: 42)

[0477] hu6VL7 (SEQ ID NO: 43)

[0478] hu6VL8 (SEQ ID NO: 44)

[0479] hu6VL9 (SEQ ID NO: 45)

[0480] hu6VL10 (SEQ ID NO: 46)

[0481] Graft(IGKV3-20*02)+Y36L,Q42K,L46G,T69A,F71Y,E79Q,V85D+D56E

[0482] hu6VL11(SEQ ID NO:47)Graft(IGKV3-20*02)+D56E

[0483] Note: The single underlined part is CDR, the double underlined part is the amino acid substitution site, and the rest is FR region, the same below.

[0484] The CDRs of the humanized mAb6 antibody are as follows:

[0485] Table 6. CDRs of mAb6 humanized antibody

[0486] Table 7. Humanized antibodies of mAb6

[0487] Note: hu6L1H1 indicates that the antibody comprises a heavy chain variable region hu6VH1 and a light chain variable region hu6VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 20, and the sequence of its light chain constant region is SEQ ID NO: 21, and so on.

[0488] Table 8. Humanized antibodies of mAb6

[0489] The above antibodies were cloned, expressed, and purified, and humanized antibodies with better activity were finally selected through protein binding experiments (Test Example 3), cell binding experiments (Test Example 2), and Biacore (Test Example 1). The amino acid sequences of the heavy and light chains of exemplary humanized antibodies are as follows:

[0490] Hu6 (also known as hu6L4H12) heavy chain (SEQ ID NO: 54):

[0491] Hu6 (also known as hu6L4H12) light chain (SEQ ID NO: 55):

[0492] Note: The underlined part in the sequence is the variable region, and the italic part is the constant region.

[0493] Example 4. Preparation of anti-DLL3-CD3 bispecific antibodies

[0494] 4.1 CD3 Antibodies Used in This Disclosure

[0495] The CD3 binding portion disclosed herein can be derived from any suitable antibody. Specifically, the disclosed embodiment uses S107E, whose variable region and CDR sequences are as follows:

[0496] Table 9. CDRs of S107E

[0497] The variable region sequence of S107E is as follows:

[0498] >S107E-VH (SEQ ID NO: 62)

[0499] >S107E-VL (SEQ ID NO: 63)

[0500] Note: The underlined part is CDR.

[0501] 4.2 Structure of the DLL3-CD3 Bispecific Antibody Disclosed

[0502] The DLL3-CD3 bispecific antibody molecule disclosed herein comprises two chains, and the specific structure is as follows:

[0503] Chain 1 structure: [VL (anti-DLL3)]-Linker 1-[VH (anti-CD3)]-Linker 2-[Fc (Knob, L234A, L235A, G237A, Y349C, T366W)];

[0504] Chain 2 structure: [VL(anti-CD3)]-Linker 3-[VH(anti-DLL3)]-Linker 2-[Fc(hole,L234A,L235A,G237A,S354C,T366S,L368A,Y407V)];

[0505] The schematic diagram is shown in Figure 1.

[0506] The Fc regions of the two chains of the bispecific antibody molecule are capable of associating with each other using a knob-in-hole technique. For example, the Fc region of chain 1 has a knob structure (e.g., SEQ ID NO: 64 or SEQ ID NO: 66) according to the knob-in-hole technique, and the Fc region of chain 2 has a hole structure (e.g., SEQ ID NO: 65 or SEQ ID NO: 67) according to the knob-in-hole technique.

[0507] The relevant sequences are as follows:

[0508] >IgG1Fc(Knob,L234A / L235A / S354C / T366W)(SEQ ID NO: 64)

[0509] >IgG1Fc(Hole,L234A,L235A,Y349C,T366S,L368A,Y407V)(SEQ ID NO: 65)

[0510] >Fc Knob (L234A, L235A, G237A, Y349C, T366W) (SEQ ID NO: 66)

[0511] >Fc hole(L234A, L235A, G237A, S354C, T366S, L368A, Y407V) (SEQ ID NO: 67)

[0512] >Linker 1 (SEQ ID NO: 68)

[0513] >Linker 2 (SEQ ID NO: 69)

[0514] >Linker 3 (SEQ ID NO: 70)

[0515] Based on the amino acid sequence of the mAb6 humanized antibody, the following bispecific antibody was constructed: hu6(D56E)-S107E-diabody

[0516] Chain 1: hu6-VL(D56E)-Linker 1-S107E-Linker 2-VH-Fc knob; (SEQ ID NO: 71)

[0517] Chain 2: CD3 VL-Linker 3-hu6 VH-Linker 2-Fc hole; (SEQ ID NO: 72)

[0518] Note: hu6(D56E)-S107E-diabody indicates that this molecule uses the variable region of the hu6(D56E) humanized antibody as the DLL3 binding domain, the variable region of S107E as the CD3 binding domain, and diabody as the molecular structure.

[0519] >Chain 1 (SEQ ID NO: 71)

[0520] >Chain 2 (SEQ ID NO: 72)

[0521] The control molecule AMG-75 used in this disclosure was prepared with reference to WO2017021349. The sequence of the control molecule is as follows:

[0522] AMG-757 (SEQ ID NO: 73):

[0523] Test Case

[0524] Test Example 1: Biacore Antibody Affinity Determination

[0525] The test antibody was affinity-captured for 18 seconds using a Protein A biosensor chip (Cat. #29127556, Cytiva). Antigens (human DLL3 (ACRO, DLL3-H52H4), cynomolgus macaque DLL3 (KACTUS, DLL-RM103), mouse DLL3 (KACTUS, DLL-MM103), human CD3D & 3E (ACRO, CDD-H52W1), and monkey CD3D & 3E (ACRO, CDD-C52W4)) were then passed over the chip surface for 180 seconds, followed by a 600-second dissociation period. Binding and dissociation curves were obtained using a Biacore 8K (Cytiva) instrument for real-time signal monitoring. After each experimental dissociation cycle, the chip was washed and regenerated with 10 mM glycine-HCl solution (pH 1.5, Cat. #BR-1003-54, Cytiva). A 1:1 model was used for data fitting. The results are shown in Tables 10 and 11.

[0526] Table 10. Affinity of antibodies to human DLL3 (based on clone 6)

[0527] Table 11. Affinity of the bispecific antibody hu6(D56E)-S107E-diabody to antigens from different species

[0528] The results showed that the humanized antibodies and chimeric antibodies disclosed herein that specifically bind to DLL3 have good affinity with human DLL3; the bispecific antibodies have good affinity with both human and cynomolgus monkey DLL3 and CD3.

[0529] Test Example 2: FACS binding experiment of DLL3 cells

[0530] The DLL3-expressing small cell lung cancer cell lines H1184 (ATCC, catalog number CRL-5858), DLL3 / H82, DLL3 / SHP77, hDLL3 / CHO-s, cynoDLL3 / CHO-s cells, and CD3-expressing Jurkat cells (ATCC, TIB-152) were prepared with FACS buffer (1% BSA + pH 7.4 PBS) to a concentration of 1×10 6 / mL cell suspension, 100μL / well was added to a 96-well round-bottom plate. Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of the test antibody, 100μL / well. Incubate in a 4℃ refrigerator in the dark for 1 hour. After washing three times by centrifugation at 300g, add working concentrations of APC anti-human IgG Fc (BioLegend, 410712) or PE F(ab')2-goat anti-human IgG (invitrogen, H10104) and incubate in a 4℃ refrigerator in the dark for 40 minutes. After washing three times by centrifugation at 300g, the geometric mean fluorescence intensity was detected on an Invitrogen flow cytometer to calculate the binding EC value of the antibody to cells expressing DLL3. 50 The results are shown in Tables 12 and 13.

[0531] Table 12. Binding activity of antibodies to H1184 cells expressing human DLL3

[0532] Table 13. Binding activity of bispecific antibodies to cells

[0533] The results showed that the anti-DLL3 humanized antibody disclosed herein has good binding ability to cells expressing human DLL3; the bispecific antibody has good binding ability to cells expressing human and cynomolgus monkey DLL3 and CD3.

[0534] Test Example 3: ELISA binding assay for DLL3 protein levels

[0535] Coat the plate with streptavidin (abcam, ab136200, 1 μg / ml) at 100 μL / well at 4°C overnight. Wash the plate three times with 250 μL / well of PBST solution (PBS containing 0.1% Tween 20). Block the plate with 5% milk at 250 μL / well at 37°C for 2 hours. Wash the plate three times with 250 μL / well of PBST solution. Add biotinylated DLL3 antigen (1 μg / mL) (SEQ ID NO: 9) and incubate at 37°C for 1 hour. Wash the plate three times with 250 μL / well of PBST solution. Prepare the antibody (maximum concentration 400 nM, 4-fold serial dilutions) and incubate at 37°C for 1 hour. Wash the plate six times with 250 μL / well of PBST solution. Add 100 μL / well of human IgG (H+L)-HRP (Jackson, 109-035-003, 1:4000 dilution) at a working concentration and incubate at 37°C for 1 hour. Wash the plate six times with 250 μL / well of PBST solution. Add 100 μL / well of TMB (KPL, 5120-0077) colorimetric solution and develop at room temperature for 5-10 minutes. Stop color development by adding 100 μL / well of 1 M H₂SO₄. Read the plate at 450 nm using a microplate reader (Molecular Devices, VERSA max).

[0536] Table 14. Binding activity of bispecific antibodies to human DLL3 protein

[0537] The results showed that the bispecific antibody disclosed herein has good affinity for human DLL3 protein.

[0538] Test Example 4: ELISA binding assay for DLL1 and DLL4 proteins

[0539] Coat the plate with DLL1 (ACRO, DL1-H52H8, 1 μg / mL) and DLL4 (ACRO, DL4-H5227, 1 μg / mL) at 100 μL / well at 4°C overnight. Wash the plate three times with PBST solution (PBS containing 0.1% Tween 20) at 250 μL / well. Block the plate with 5% milk at 250 μL / well at 37°C for 2 hours. Wash the plate three times with PBST solution at 250 μL / well. Add the antibody to be detected (maximum concentration 100 nM, 4-fold serial dilution) and incubate at 37°C for 1 hour. Wash the plate six times with PBST solution at 250 μL / well. Add the working concentration of human IgG (H+L)-HRP (Jackson, 109-035-003, 1:4000 dilution) at 100 μL / well and incubate at 37°C for 1 hour. Wash the plate six times with 250 μL / well of PBST solution. Add 100 μL / well of TMB (KPL, 5120-0077) colorimetric solution and develop at room temperature for 5-10 minutes. Add 100 μL / well of 1 M H₂SO₄ to stop color development. Read the plate at 450 nm using a microplate reader (Molecular Devices, VERSA max). The results are shown in Figures 2A-2B.

[0540] The results showed that the bispecific antibodies disclosed herein did not detectably bind to human DLL1 and DLL4 proteins.

[0541] Test Example 5. Cytotoxicity of the Bispecific Antibody of the Present Disclosure

[0542] This test example investigated the cytotoxic activity of bispecific antibodies as T cell engager molecules against tumor cells. SHP77, H1184, and H460 stably transfected cell lines expressing LUC-GFP were used as target cells to detect the target-specific cytotoxic activity of the bispecific antibodies disclosed herein.

[0543] SHP77 / lucG cells were cultured in 1640+10% FBS+10μg / mL puromycin complete medium and passaged 2-3 times a week. H1184 / lucG cells were cultured in 1640+5% FBS+10μg / mL puromycin complete medium and passaged once a week. H460 / lucG cells were cultured in 1640+10% FBS complete medium and passaged 2-3 times a week. After thawing, cryopreserved PBMCs (Miaoshun Bio) were resuspended in 1640+10% FBS complete medium and placed in a T75 culture flask for 4 hours (density of 2E6 cells / mL). After collecting and centrifuging, PBMCs were resuspended in 1640+10% FBS, counted, and the cell number was adjusted to 1.5E6 cells / mL. The above PBMC suspension and target cell suspension were mixed in equal volumes and 100μL was added to each well to ensure an E:T ratio of 10:1. A separate PBMC-only group was established. The PBMC suspension and 1640 complete medium with 10% FBS were mixed in equal volumes. The antibody was diluted in 1640 complete medium with 10% FBS to a starting concentration of 600 nM (6x the final concentration). Five-fold dilutions were added to each well across nine dose points. The treated cells were incubated at 37°C, 5% CO₂ in a humidified incubator for 48 hours. The plate was removed and centrifuged at 1000 rpm for 3 minutes. 50 μL of the supernatant was transferred to a new 3788 plate for cytokine analysis and stored at -20°C. 50 μL of ONE-Glo™ Luciferase (Promega, E6120) was added to the plate. After incubation at room temperature for 5 minutes, luminescence was measured using a Vendor assay. The cytotoxicity of the antibody at different concentrations was calculated. Dose-response curves were generated using Graphpad Prism 8.0 software based on the logarithmic concentration of the antibody and the signal value, and the IC₅₀ value for antibody-mediated cytotoxicity was calculated. Wells containing only target cells and PBMC without antibody were set as 0% inhibition, and the maximum inhibition rate Imax of the antibody was calculated. The results are shown in Table 15 and Figure 3.

[0544] Table 15. Cytotoxic activity of bispecific antibody hu6(D56E)-S107E-diabody against different target cells

[0545] The results showed that the bispecific antibody disclosed herein had a strong killing effect on DLL3-expressing cells SHP77 and H1184; but had no killing effect on H460 cells that did not express DLL3 ( FIG. 3 ).

[0546] Test Example 6. In vitro T cell activation activity of the bispecific antibodies disclosed herein

[0547] H82 / DLL3 cells were cultured in 1640+10% FBS complete medium and passaged 2-3 times a week at a passage ratio of about 1:3. H460 cells were cultured in 1640+10% FBS complete medium and passaged 2-3 times a week at a passage ratio of about 1:10. TM NFAT cells (InvivoGen) were cultured in 1640+10% FBS+100 μg / ml zeocin (InvivoGen, ant-zn-1) complete medium and passaged 2-3 times a week at a passage ratio of about 1:10. TM NFAT cells were centrifuged at 1000 rpm for 3 minutes, resuspended, counted, and the cell count was adjusted to 1E6 cells / mL. Target H82 / DLL3 cells were collected, centrifuged at 1000 rpm for 3 minutes, resuspended, counted, and the cell count was adjusted to 2E5 cells / mL. H82 / DLL3 and H460 cells were collected, centrifuged at 1000 rpm for 3 minutes, resuspended, counted, and the cell count was adjusted to 4E5 cells / mL. TM NFAT and target cells were mixed in equal volumes and 100 μL was added to each well, ensuring an E:T ratio of 5:1 for H82 / DLL3 cells and 2.5:1 for H82 / DLL3 and H460 cells. Antibody was diluted in 1640 medium with 10% FBS to a starting concentration of 600 nM (6× final concentration). Five-fold dilutions were performed across nine dose points, with 20 μL added to each well. Treated cells were incubated at 37°C, 5% CO2 for 5 hours. QUANTI-Luc was dissolved in 25 mL of ddH2O. TM Gold reagent (Invivogen, rep-qlcg5). Add 50 μL QUANTI-Luc TM Gold, incubated at room temperature for 5 minutes, luminescence was detected by Vendor, and the antibody activity against Jurkat Lucia at different concentrations was calculated. TM Activation of NFAT cells. Graphpad Prism 8.0 software was used to generate graphs, and the results are shown in Figures 4A to 4B and Table 16.

[0548] The formula for activation multiple is N = fluorescence reading of the antibody to be tested / fluorescence reading of the negative well

[0549] Table 16. Results of T cell activation by bispecific antibodies

[0550] The results showed that the bispecific antibody disclosed herein can activate T cells only in the presence of DLL3 / H82 cells that express DLL3; it will not activate T cells in the presence of negative cells H460 that do not express DLL3.

[0551] Test Example 7. Cytokine Release Level of the Bispecific Antibody of the Disclosure

[0552] 1. Cytokine IFNγ Release Levels of the Bispecific Antibodies of the Disclosure

[0553] The HTRF method was used to detect the secretion of cytokine IFNγ in PBMCs stimulated by DLL3 / CD3 dual antibodies in the presence of H1184 cells.

[0554] Remove the supernatant collected in the cell killing experiment (Test Example 5) and stored in a -20°C refrigerator, thaw at room temperature, and shake to mix. Dissolve the IFNγ standard powder in sterile water and dilute the standard to the required concentration using 1640+10% FBS medium. Remove the kit for detecting IFNγ (CISBIO, 62HIFNGPEG), equilibrate the kit to room temperature, and dilute the two detection antibodies in the kit with detection buffer (20-fold dilution). Take 16 μL of cell supernatant (10-fold dilution for IFNγ detection) and IFNγ standard into a 96-well plate, add 4 μL of the diluted corresponding detection antibody; shake to mix, centrifuge at 1000 rpm for 1 minute, and incubate overnight at room temperature. Remove the overnight incubation sample, centrifuge at 1000 rpm for 1 minute, and read the absorbance values ​​at 665 nm and 620 nm using a PHERAstar multi-function microplate reader. Data were analyzed using Graphpad Prism 8, and the results are shown in Figure 5A.

[0555] The results showed that the bispecific antibodies of the present disclosure released very low levels of IFNγ.

[0556] 2. Cytokine IL-6 Release Levels of the Bispecific Antibodies of the Disclosure

[0557] ELISA was used to detect the secretion of cytokine IL-6 in PBMCs stimulated by the presence of DLL3 / CD3 dual antibodies in the presence of H1184 cells.

[0558] The supernatant collected from the cell killing experiment (Test Example 5) and stored at -20°C was removed and thawed at room temperature. The sample was then mixed by vortexing and diluted 5-fold with sample diluent for later use. Prior to cytokine detection, an IL-6 ELISA kit (Xinbosheng Biotechnology Co., Ltd., EHC007.96) was taken out and equilibrated at room temperature. The IL-6 standard was diluted and the OD450 value was measured according to the instructions. The data was analyzed using Graphpad Prism 8. The results are shown in Figure 5B.

[0559] The results showed that the level of IL6 released by the bispecific antibody of the present disclosure was very low, indicating that the bispecific antibody of the present disclosure has better safety.

[0560] In vivo activity evaluation

[0561] Test Example 8. Efficacy of the bispecific antibody disclosed herein in the SHP77 subcutaneous transplant tumor model

[0562] The present disclosure uses severe combined immunodeficient NOG mice to be inoculated with SHP-77 cells, and then intraperitoneally injected with human PBMCs. After tumor formation, the mice are divided into groups for drug administration.

[0563] Female NOG mice, weighing 15–17 g, were purchased from Vital River. SHP-77 cells were obtained from ATCC. Human PBMCs were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., catalog number A10S115034.

[0564] SHP-77 cells (4×10 6 hPBMCs were thawed on the day of inoculation. On the second day of inoculation, 5×10 hPBMCs were injected intraperitoneally into each mouse. 6 8 days after inoculation, when the tumor volume is 120 mm 3 After weight loss and tumor size were eliminated, mice were randomly divided into five groups of eight mice each based on tumor volume. Starting on the same day, equimolar amounts of antibodies were administered, as shown in Table 32. The corresponding equimolar amounts of antibodies were intraperitoneally injected once every five days for a total of three doses until day 15. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0565] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0566] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where 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.

[0567] Tumor inhibition rate TGI (%) = 100 - T / C (%).

[0568] Table 17. Efficacy of Antibodies in SHP77 Subcutaneous Xenograft Tumor Model (Day 15 after Administration)

[0569] The results showed that the bispecific antibody disclosed herein can significantly inhibit the growth of SHP-77 transplanted tumors.

[0570] Test Example 9. Efficacy of the bispecific antibody disclosed herein in the H1184 subcutaneous transplant tumor model

[0571] The present disclosure uses severe combined immunodeficient NOG mice to be inoculated with H1184 cells, and then intraperitoneally injected with human PBMCs. After tumor formation, the mice are divided into groups and given the drug.

[0572] Female NOG mice, weighing 15–17 g, were purchased from Vital River. H1184 cells were obtained from ATCC. Human PBMCs were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., catalog number A10S115034.

[0573] H1184 cells (3.5×10 6 200 μL of 50% MatriGel per mouse was inoculated subcutaneously in the right flank of NOG mice. On the 11th day after inoculation, 8 frozen hPBMCs (ID#A10S115034) were revived. On the 12th day after inoculation, 5×10 human PBMCs were injected intraperitoneally into each mouse. 6 / 100μL cell suspension. 18 days after inoculation, when the tumor volume is ~140mm 3 After weight loss and tumor size were determined, mice were randomly divided into groups of 8 mice based on tumor volume. Starting on the same day, equimolar doses of the antibodies were administered, as shown in Table 33. Antibodies were administered via intraperitoneal injection for a total of 4 doses, twice weekly. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0574] Excel statistical software was used to record data: mean value was calculated as avg; SD value was calculated as STDEV; SEM value was calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism software was used to draw graphs, and two-way ANOVA, one-way ANOVA, and t-test were used to perform statistical analysis of the data.

[0575] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0576] Relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where 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.

[0577] Tumor inhibition rate TGI (%) = 100 - T / C (%).

[0578] Table 18. Antibody efficacy in H1184 subcutaneous transplant tumor model (15 days after administration)

[0579] The results showed that the bispecific antibody disclosed herein can significantly inhibit the growth of H1184 transplanted tumors.

[0580] II. Preparation Example - Bispecific Antibody Preparation Specific for Binding to DLL3 and CD3

[0581] SEC size exclusion chromatography:

[0582] 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.

[0583] 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.

[0584] SEC measurement instrument: Agilent HPLC 1260;

[0585] Column: Waters, BioResolve TM SEC mAb 2.5μm 7.8×300mm Column

[0586] NR-CE capillary gel electrophoresis:

[0587] 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.

[0588] 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.

[0589] CE measurement instrument: Beckman capillary electrophoresis instrument, model PA800 plus

[0590] iCIEF whole-column imaging capillary isoelectric focusing electrophoresis:

[0591] A technique for separating proteins based on their isoelectric points (pI).

[0592] 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.

[0593] Instrument used for iCIEF determination: simple protein, model Muarice.

[0594] IEC ion exchange chromatography:

[0595] 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.

[0596] 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.

[0597] IEC measurement instrument: Agilent HPLC 1260.

[0598] Osmolality determination:

[0599] 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.

[0600] Instrument for osmotic pressure measurement: Loser, model OM815.

[0601] protein

[0602] The protein used in the following examples is a bispecific antibody protein (hu6(D56E)-S107E-diabody, hereinafter referred to as "protein") that specifically binds to DLL3 and CD3.

[0603] The concentration of the fusion protein was determined using a UV-visible spectrophotometer, model: Nano Drop oneC, with an optical path length of 1 mm.

[0604] Formulation Example 1. pH and buffer system screening

[0605] Formulations containing 70 mg / mL protein, 80 mg / mL sucrose, and 0.4 mg / mL polysorbate 80 (PS80) were prepared using the buffer system shown in Table 19. Forced degradation studies (40°C for 4 weeks) were conducted to further investigate the effects of different pH values ​​and buffer systems on protein stability, using SEC and IEC as evaluation metrics.

[0606] The results are shown in Table 19. SEC data showed that ΔSEC ranged from 0.8% to 3.2%, with the His-HCl system (pH 4.5-5.5) and the His-AA system (pH 5.0) being superior. ΔIEC data showed that the His-HCl system (pH 4.5-5.5) and the CA system (pH 5.5) were superior. Based on the ΔSEC and ΔIEC data, the His-HCl system (pH 4.5-5.5) was preferred.

[0607] Table 19. pH and buffer system screening results

[0608] Note: His-HCl stands for histidine-histidine hydrochloride; SA stands for succinic acid-sodium succinate; CA stands for citric acid-sodium citrate; His-AA stands for histidine-histidine acetate; 40℃ W4: 40℃ for 4 weeks, the same below.

[0609] Formulation Example 2. Protein Concentration Screening

[0610] Formulations containing 0.1 mg / mL ethylenediaminetetraacetic acid disodium dihydrate (containing dihydrate, manufacturer: Hunan Erkang Pharmaceutical Co., Ltd., hereinafter referred to as EDTA-2Na, CAS: 6381-92-6), 80 mg / mL sucrose, 0.4 mg / mL PS80, and the protein concentrations shown in Table 20 were prepared using 18 mM His-HCl buffer (pH 5.0). Forced degradation studies (40°C for 4 weeks) were performed on the samples to investigate the effects of different protein concentrations on protein stability, using SEC and IEC as evaluation indicators.

[0611] The results are shown in Table 20. After 4 weeks at 40°C, ΔSEC showed a slight increasing trend with increasing protein concentration, while ΔIEC showed little difference among the groups.

[0612] Table 20. Protein concentration screening results

[0613] Preparation Example 3. EDTA-2Na concentration screening

[0614] Formulations containing 100 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL PS80, and the various EDTA-2Na concentrations shown in Table 21 were prepared using 18 mM His-HCl buffer (pH 5.0). Forced degradation studies (40°C for 4 weeks) were performed to investigate the effects of different EDTA-2Na concentrations on formulation stability using SEC, NR-CE, and IEC.

[0615] The results are shown in Table 21. After 4 weeks at 40°C, ΔSEC was within the range of 0.7%-1.4%. The monomer purity of the preparation containing 0.1 mg / mL EDTA-2Na was slightly better than that of the other groups. There was no significant difference between the ΔNR-CE and ΔIEC groups.

[0616] Table 21. EDTA-2Na concentration screening results

[0617] Preparation Example 4. Screening of ion concentration of His-HCl buffer system

[0618] Formulations containing 100 mg / mL protein, 0.1 mg / mL EDTA-2Na, 80 mg / mL sucrose, and 0.4 mg / mL PS80 were prepared using His-HCl buffer (pH 5.0) with varying ion concentrations as shown in Table 22. Forced degradation studies (40°C for 4 weeks) were performed to investigate the effect of varying histidine ion concentrations on protein stability using SEC, NR-CE, and IEC.

[0619] The results are shown in Table 22. After 4 weeks at 40°C, there were no significant differences among the ΔSEC, ΔNR-CE and ΔIEC groups.

[0620] Table 22. His-HCl buffer system concentration screening results

[0621] Formulation Example 5. Sugar Concentration Screening

[0622] Formulations containing 100 mg / mL protein, 0.1 mg / mL EDTA-2Na, 0.4 mg / mL PS80, and varying sucrose concentrations as shown in Table 23 were prepared using 18 mM His-HCl buffer (pH 5.0). Forced degradation studies (40°C for 4 weeks) were performed to investigate the effects of varying sucrose concentrations on formulation stability using SEC, NR-CE, and IEC.

[0623] The results are shown in Table 23. ΔSEC was in the range of 0.7%-1.5%, and the monomer purity of the formulation containing 80 mg / mL sucrose was slightly better than that of the other groups. There was no significant difference between the ΔNR-CE and ΔIEC groups.

[0624] Table 23. Sucrose concentration screening results

[0625] Sugar is a common excipient in biopharmaceuticals. This project used sucrose as an osmotic pressure regulator and stabilizer for formulation screening, which met the formulation requirements. At a sucrose concentration of 80 mg / mL, the actual measured osmotic pressure of the formulation was 292 mOsm, approaching isotonicity. Therefore, a sucrose concentration of 80 mg / mL was the preferred concentration.

[0626] Formulation Example 6. Screening of surfactant types

[0627] Formulations containing 70 mg / mL protein, 80 mg / mL sucrose, and various surfactants, including PS80 and poloxamer 188 (P188), were prepared using 18 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 appearance, SEC, NR-CE, and iCIEF as evaluation criteria.

[0628] The results are shown in Table 24. Appearance, ΔSEC, ΔNR-CE, and ΔiCIEF data showed that after 4 weeks at 40°C, there were no significant differences in appearance and purity among formulations containing different types of surfactants.

[0629] Table 24. Surfactant concentration screening results

[0630] Formulation Example 7. Surfactant Concentration Screening

[0631] Formulations containing 100 mg / mL protein, 0.1 mg / mL EDTA-2Na, 80 mg / mL sucrose, and various PS80 concentrations as shown in Table 25 were prepared using 18 mM His-HCl buffer (pH 5.0). Forced degradation studies (40°C for 4 weeks) were performed to investigate the effects of different surfactant concentrations on protein stability, using appearance, SEC, NR-CE, and IEC as evaluation criteria.

[0632] The results are shown in Table 25. Appearance, ΔSEC, ΔNR-CE, and ΔIEC data showed that after 4 weeks at 40°C, there was no significant difference in appearance and purity among the formulations containing different concentrations of PS80.

[0633] Table 25. Surfactant concentration screening results

[0634] Preparation Example 8. Prescription Confirmation

[0635] A formulation containing 150 mg / mL protein, 0.1 mg / mL EDTA-2Na, 80 mg / mL sucrose, and 0.4 mg / mL PS80 was prepared using 18 mM His-HCl buffer (pH 5.0). A long-term stability study (6 months at 5°C) was conducted to assess protein stability using SEC and IEC.

[0636] The results are shown in Table 26. After 6 months at 5°C, there was no significant change in SEC and IEC compared with day 0, indicating that the protein had good stability under this protein concentration condition.

[0637] Table 26. Long-term stability results of formulations with a protein concentration of 150 mg / mL

[0638] Preparation Example 9. Prescription Confirmation

[0639] A formulation of 100 mg / mL protein, 0.1 mg / mL EDTA-2Na, 0.4 mg / mL PS80, and 80 mg / mL sucrose was prepared using 18 mM His-HCl buffer (pH 5.0). Long-term stability studies were conducted using SEC, NR-CE, and IEC as evaluation criteria.

[0640] The results are shown in Table 27. SEC, NR-CE, and IEC data showed that there was no significant change in the purity of the preparation after storage at 5°C for 12 months.

[0641] Table 27. Long-term stability results

[0642] Note: 5℃, M3 / M12: 3 months / 12 months at 5℃.

[0643] Preparation Example 10. Prescription Confirmation

[0644] A formulation of 5 mg / mL protein, 0.1 mg / mL EDTA-2Na, 0.4 mg / mL PS80, and 80 mg / mL sucrose was prepared using 18 mM His-HCl buffer (pH 5.0). Long-term stability studies were conducted on the samples, using SEC and IEC as evaluation indicators.

[0645] The results are shown in Table 28. SEC and IEC data showed that there was no significant change in the purity of the preparation after storage at 5°C for 6 months.

[0646] Table 28. Long-term stability results

[0647] Note: 5℃, M1 / M6: 1 month / 6 months at 5℃.

[0648] Although the above invention has been described in detail with the aid of the accompanying drawings and examples for clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition comprising a bispecific antibody that specifically binds to DLL3 and CD3 and a buffer, wherein: The bispecific antibody that specifically binds to DLL3 and CD3 comprises a first chain having a structure shown in Formula I and a second chain having a structure shown in Formula II, Formula I: [DLL3-VL]-[Linker 1]-[CD3-VH]-[Linker 2]-[Fc1], Formula II: [CD3-VL]-[Linker 3]-[DLL3-VH]-[Linker 2]-[Fc2], The DLL3-VH comprises a DLL3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a DLL3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a DLL3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 16; and the DLL3-VL has: a DLL3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 17, a DLL3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a DLL3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and The CD3-VH comprises a CD3-HCDR1 comprising an amino acid sequence of SEQ ID NO: 56, a CD3-HCDR2 comprising an amino acid sequence of SEQ ID NO: 57, and a CD3-HCDR3 comprising an amino acid sequence of SEQ ID NO: 58; and the CD3-VL has: a CD3-LCDR1 comprising an amino acid sequence of SEQ ID NO: 59, a CD3-LCDR2 comprising an amino acid sequence of SEQ ID NO: 60, and a CD3-LCDR3 comprising an amino acid sequence of SEQ ID NO: 61; Wherein: the structures shown in Formula I and Formula II are arranged from the N-terminus to the C-terminus; the linker 1, linker 2 and linker 3 are identical or different peptide linkers; the Fc1 and the Fc2 are Fc region structural sequences that can associate with each other using a knob-and-hole technique; 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, histidine-histidine acetate buffer or citric acid-sodium citrate 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 4.5 to 6.0; preferably, the pH of the pharmaceutical composition is 4.5 to 5.5; more preferably, the pH of the pharmaceutical composition is 4.8 to 5.2; most preferably, the pH of the pharmaceutical composition is about 5.

0.

3. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL to 250 mg / mL; Preferably, the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL to 150 mg / mL; Further preferably, the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 70 mg / mL to 150 mg / mL; More preferably, the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 80 mg / mL to 120 mg / mL; Most preferably, the concentration of the bispecific antibody that specifically binds DLL3 and CD3 is about 100 mg / mL.

4. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL to 20 mg / mL; Preferably, the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg / mL to 10 mg / mL; More preferably, the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 4 mg / mL to 6 mg / mL; Most preferably, the concentration of the bispecific antibody that specifically binds DLL3 and CD3 is about 5 mg / mL.

5. The pharmaceutical composition according to any one of claims 1 to 4, 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.

6. The pharmaceutical composition according to claim 5, wherein the concentration of polysorbate 80 is 0.01 mg / mL to 1.0 mg / mL; preferably, the concentration of polysorbate 80 is 0.1 mg / mL to 1.0 mg / mL; further preferably, the concentration of polysorbate 80 is 0.2 mg / mL to 0.6 mg / mL; more preferably, the concentration of polysorbate 80 is 0.3 mg / mL to 0.5 mg / mL; most preferably, the concentration of polysorbate 80 is about 0.4 mg / mL.

7. The pharmaceutical composition according to claim 5, wherein the concentration of poloxamer 188 is 0.5 mg / mL to 5.0 mg / mL; preferably, the concentration of poloxamer 188 is 1 mg / mL to 3 mg / mL; more preferably, the concentration of poloxamer 188 is 1.5 mg / mL to 2.5 mg / mL; most preferably, the concentration of poloxamer 188 is about 2.0 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition comprises sugar; preferably, the sugar is sucrose, trehalose, mannitol or sorbitol; more preferably, the sugar is sucrose.

9. The pharmaceutical composition of claim 8, wherein the sugar concentration is 10 mg / mL to 120 mg / mL; preferably, the sugar concentration is 30 mg / mL to 100 mg / mL; more preferably, the sugar concentration is 64 mg / mL to 96 mg / mL; most preferably, the sugar concentration is about 80 mg / mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the concentration of the buffer is 5 mM to 100 mM; preferably, the concentration of the buffer is 10 mM to 50 mM; further preferably, the concentration of the buffer is 10 mM to 30 mM; more preferably, the concentration of the buffer is 15 mM to 25 mM; most preferably, the concentration of the buffer is about 18 mM.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition further comprises an excipient; preferably, the excipient is disodium ethylenediaminetetraacetic acid dihydrate, DTPA, 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.

12. The pharmaceutical composition according to claim 11, 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 0.5 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.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein in the bispecific antibody that specifically binds to DLL3 and CD3: The DLL3-VH comprises the amino acid sequence of SEQ ID NO: 33, and the DLL3-VL comprises the amino acid sequence of SEQ ID NO: 42; and The CD3-VH comprises the amino acid sequence of SEQ ID NO:62, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:

63.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the Fc1 in the bispecific antibody that specifically binds to DLL3 and CD3 has a protrusion structure according to the knob-and-mortar technique, and the Fc2 has a hole structure according to the knob-and-mortar technique; Preferably, the amino acid sequence of Fc1 is shown as SEQ ID NO: 66; and the amino acid sequence of Fc2 is shown as SEQ ID NO:

67.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the linker 1, linker 2 and linker 3 in the bispecific antibody that specifically binds to DLL3 and CD3 are the same or different, and are each independently selected from SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO:

70.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the bispecific antibody specifically binds to DLL3 and CD3, wherein The amino acid sequence of Formula I is shown in SEQ ID NO:71, and the amino acid sequence of Formula II is shown in SEQ ID NO:

72.

17. The pharmaceutical composition according to claim 1, comprising the following components: (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.01 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188, (c) 10 mg / mL to 120 mg / mL of sugar, (d) 0.01 mg / mL to 1 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition is 4.5 to 6.0; Preferably, the pharmaceutical composition comprises the following components: (a) 1 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or 1 mg / mL to 3 mg / mL of poloxamer 188, (c) 30 mg / mL to 100 mg / mL of sucrose, (d) 0.01 mg / mL to 1 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer or citric acid-sodium citrate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 5.5; Further preferably, the pharmaceutical composition comprises the following components: (a) 70 mg / mL to 150 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.2 mg / mL to 0.6 mg / mL of polysorbate 80 or 1 mg / mL to 3 mg / mL of poloxamer 188, (c) 64 mg / mL to 96 mg / mL of sucrose, (d) 0.01 mg / mL to 0.5 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) 10 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 4.5 to 5.5; More preferably, the pharmaceutical composition comprises the following components: (a) 80 mg / mL to 120 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.3 mg / mL to 0.5 mg / mL of polysorbate 80 or 1.5 mg / mL to 2.5 mg / mL of poloxamer 188, (c) 64 mg / mL to 96 mg / mL of sucrose, (d) 0.08 mg / mL to 0.12 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 4.8 to 5.2: Most preferably, the pharmaceutical composition comprises the following components: (a) about 100 mg / mL of the bispecific antibody that specifically binds DLL3 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 disodium ethylenediaminetetraacetate dihydrate, and (e) about 18 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.

0.

18. The pharmaceutical composition according to claim 1, comprising the following components: (a) 1 mg / mL to 20 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.01 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188, (c) 10 mg / mL to 120 mg / mL of sugar, (d) 0.01 mg / mL to 1 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition is 4.5 to 6.0; Preferably, the pharmaceutical composition comprises the following components: (a) 1 mg / mL to 10 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.1 mg / mL to 1.0 mg / mL of polysorbate 80 or 0.5 mg / mL to 5 mg / mL of poloxamer 188, (c) 30 mg / mL to 100 mg / mL of sucrose, (d) 0.01 mg / mL to 1 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer or citric acid-sodium citrate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 5.5; Further preferably, the pharmaceutical composition comprises the following components: (a) 1 mg / mL to 10 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.1 mg / mL to 1.0 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 disodium ethylenediaminetetraacetate dihydrate, and (e) 10 mM to 50 mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 4.5 to 5.5; More preferably, the pharmaceutical composition comprises the following components: (a) 4 mg / mL to 6 mg / mL of the bispecific antibody that specifically binds to DLL3 and CD3, (b) 0.3 mg / mL to 0.5 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 disodium ethylenediaminetetraacetate dihydrate, and (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer, the pH of the pharmaceutical composition is 4.8 to 5.2; Most preferably, the pharmaceutical composition comprises the following components: (a) about 5 mg / m of the bispecific antibody that specifically binds to DLL3 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 disodium ethylenediaminetetraacetate dihydrate, and (e) about 18 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.

0.

19. The pharmaceutical composition according to any one of claims 1 to 18, which is a subcutaneous injection preparation, an intravenous injection preparation, an intraperitoneal injection preparation or an intramuscular injection preparation; preferably a subcutaneous injection preparation.

20. A method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 18.

21. A lyophilized preparation obtained by the method of claim 20.

22. Use of the pharmaceutical composition according to any one of claims 1 to 19, or the lyophilized preparation according to claim 21, in the preparation of a medicament for treating a tumor or cancer; preferably, wherein the tumor or cancer is selected from: Lung cancer, small cell lung cancer, large cell lung cancer, head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, large cell lung cancer, triple-negative breast cancer, and lymphoma.

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