Pharmaceutical composition comprising Anti-DLL3 antibody-drug conjugate

Abstract

The present disclosure relates to a pharmaceutical composition comprising an anti-DLL3 antibody-drug conjugate. Particularly, the present disclosure relates to use of the pharmaceutical composition comprising the anti-DLL3 antibody-drug conjugate in the preparation of a drug for treating or preventing diseases.

Description

A pharmaceutical composition comprising an anti-DLL3 antibody drug conjugate

[0001] This application claims priority to Chinese patent application CN202411179425.3, filed on August 27, 2024. Technical Field

[0002] This disclosure pertains to the field of pharmaceutical formulations, specifically relating to a pharmaceutical composition comprising an anti-DLL3 antibody-drug conjugate. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] Small cell lung cancer (SCLC) is a relatively malignant type of lung cancer, accounting for 10% to 15% of all lung cancer cases. SCLC tumors grow rapidly, are prone to metastasis, and have a 5-year survival rate of less than 7%. Treatment for SCLC often involves platinum-based chemotherapy combined with etoposide. SCLC patients initially respond well to chemotherapy, but resistance and relapse are highly likely. Recent immunotherapies, such as PD-L1 and PD-1 antibodies, have shown some efficacy in SCLC patients, but the response rate is approximately 15%. Currently, no specific targeted therapy has been developed.

[0005] DLL3 is a ligand that inhibits Notch. Under normal conditions, DLL3 resides on the Golgi apparatus. In cancer cells (such as small cell lung cancer cells), DLL3 translocates to the cell surface and binds to Notch in a cis-regulatory manner, hindering cell-cell binding and Notch endocytosis within target cells, thereby inhibiting the Notch signaling pathway and promoting tumor cell growth. DLL3 is primarily expressed in neuroendocrine tumors, including SCLC, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, small cell bladder cancer, glioma multiforme, metastatic castration prostate cancer, and melanoma. Especially in SCLC, DLL3 is expressed in over 80% of SCLC cases, while it is not expressed in normal lung cancer tissue or adjacent tissues. This differential expression makes DLL3 a highly promising therapeutic target for SCLC.

[0006] Ecinotecan toxoid is a camptothecin derivative that inhibits topoisomerase I, selectively inhibiting DNA replication in proliferating tumor cells. Furthermore, eccinotecan toxoid exhibits excellent membrane permeability, allowing it to penetrate killed cancer cells and continue killing adjacent cancer cells, demonstrating a clear bystander effect in clinical practice.

[0007] Antibody-drug conjugates (ADCs) are obtained by linking antibodies to biologically active drugs via linkers. ADCs fully utilize the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of drugs (such as cytotoxic agents), while avoiding the drawbacks of low efficacy of antibodies and excessive toxic side effects of drugs. Compared with traditional chemotherapy drugs, antibody-drug conjugates can more precisely kill tumor cells and reduce the impact on normal cells.

[0008] ADCs have a more complex heterogeneous structure than antibodies, thus posing a greater challenge to ADC formulations for therapeutic purposes. Summary of the Invention

[0009] This disclosure provides a pharmaceutical composition comprising an anti-DLL3 antibody-drug conjugate. The pharmaceutical composition exhibits good therapeutic activity, safety, pharmacokinetic properties, and drug-likeness (e.g., stability).

[0010] In some embodiments, this disclosure provides a pharmaceutical composition comprising an anti-DLL3 antibody-drug conjugate and a buffer, wherein:

[0011] The anti-DLL3 antibody-drug conjugate has the following structure:

[0012] in:

[0013] n is between 1 and 10;

[0014] Pc is an anti-DLL3 antibody, which contains a heavy chain variable region and a light chain variable region, wherein:

[0015] The heavy chain variable regions HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, and the light chain variable regions LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

[0016] The buffer is a histidine buffer or a succinate buffer.

[0017] In some embodiments, the pharmaceutical composition as described above, wherein the buffer is a histidine-histidine hydrochloride buffer or a succinate-sodium succinate buffer.

[0018] In some embodiments, such as the pharmaceutical composition described in any of the preceding embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0019] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, and the amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively.

[0020] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:55.

[0021] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:50, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:55.

[0022] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-DLL3 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61.

[0023] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the anti-DLL3 antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 60, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 61.

[0024] In some embodiments, such as the pharmaceutical composition described in any of the preceding embodiments, wherein n is the average number of pharmaceutical modules per anti-DLL3 antibody, and may be an integer or a decimal. In some embodiments, n is 1-10, or 2-10, or 3-10, or 4-10, or 5-10, or 6-10, or 7-10, or 8-10, or 1-9, or 2-9, or 3-9, or 4-9, or 5-9, or 6-9, or 7-9, or 1-8, or 2-8, or 3-8, or 4-8, or 5-8, or 6-8, or 1-7, or 2-7, or 3-7, or 4-7, or 5-7, or 1-6, or 2-6, or 3-6, or 4-6, or 1-5, or 2-5, or 3-5, or 1-4, or 2-4, or 1-3. In some implementations, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0025] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein n is 3 to 8. In some embodiments, n is 4 to 8. In some embodiments, n is 6 to 8. In some embodiments, n is about 8. In some embodiments, n is 8.

[0026] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the anti-DLL3 antibody-drug conjugate is from 1 mg / mL to 50 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is from 5 mg / mL to 30 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is from 15 mg / mL to 25 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is from 16 mg / mL to 24 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is from 18 mg / mL to 22 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is about 5 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is about 20 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is about 30 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL, or any range between these values. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is 5 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is 20 mg / mL. In some embodiments, the concentration of the anti-DLL3 antibody-drug conjugate is 30 mg / mL.

[0027] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the buffer is from 5 mM to 35 mM. In some embodiments, the concentration of the buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the buffer is from 5 mM to 15 mM. In some embodiments, the concentration of the buffer is from 10 mM to 30 mM. In some embodiments, the concentration of the buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the buffer is from 9 mM to 11 mM. In some embodiments, the concentration of the buffer is from 7 mM to 11 mM. In some embodiments, the concentration of the buffer is from 8 mM to 10 mM. In some embodiments, the concentration of the buffer is from 24 mM to 36 mM. In some embodiments, the concentration of the buffer is 25 mM to 35 mM. In some embodiments, the concentration of the buffer is 27 mM to 33 mM. In some embodiments, the concentration of the buffer is 22 mM to 33 mM. In some embodiments, the concentration of the buffer is 25 mM to 30 mM. In some embodiments, the concentration of the buffer is about 5 mM. In some embodiments, the concentration of the buffer is about 9.2 mM. In some embodiments, the concentration of the buffer is about 10 mM. In some embodiments, the concentration of the buffer is about 20 mM. In some embodiments, the concentration of the buffer is about 27.8 mM. In some embodiments, the concentration of the buffer is about 30 mM. In some embodiments, the concentration of the buffer is 1 mM, 5 mM, 7 mM, 8 mM, 9 mM, 9.2 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 22 mM, 24 mM, 25 mM, 27 mM, 27.8 mM, 30 mM, 33 mM, 35 mM, 36 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the buffer is 5 mM. In some embodiments, the concentration of the buffer is 9.2 mM. In some embodiments, the concentration of the buffer is 10 mM. In some embodiments, the concentration of the buffer is 20 mM. In some embodiments, the concentration of the buffer is 27.8 mM. In some embodiments, the concentration of the buffer is 30 mM.

[0028] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the histidine-histidine hydrochloride buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 35 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 15 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 10 mM to 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 9 mM to 11 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 7 mM to 11 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 8 mM to 10 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 24 mM to 36 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 25 mM to 35 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 27 mM to 33 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 22 mM to 33 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 25 mM to 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 5 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 9.2 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 10 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 27.8 mM. 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 1 mM, 5 mM, 7 mM, 8 mM, 9 mM, 9.2 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 22 mM, 24 mM, 25 mM, 27 mM, 27.8 mM, 30 mM, 33 mM, 35 mM, 36 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 5 mM.In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 9.2 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 10 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 27.8 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 30 mM.

[0029] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments has a pH of 4.5 to 8.0. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.1. In some embodiments, the pharmaceutical composition has a pH of 5.2 to 5.8. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.0. In some embodiments, the pharmaceutical composition has a pH of 5.5 to 6.0. In some embodiments, the pharmaceutical composition has a pH of 5.1 to 6.1. In some embodiments, the pharmaceutical composition has a pH of about 5.0. In some embodiments, the pharmaceutical composition has a pH of about 5.1. In some embodiments, the pharmaceutical composition has a pH of about 5.5. In some embodiments, the pharmaceutical composition has a pH of about 5.6. In some embodiments, the pharmaceutical composition has a pH of about 6.0. In some embodiments, the pharmaceutical composition has a pH of about 6.1. In some embodiments, the pH of the pharmaceutical composition 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, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, 7.5, or 8.0, or any range between these values. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.1. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.6. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.1. When point values ​​are mentioned in this disclosure, it should be understood that the point value includes a range of error. This range of error is due to factors such as laboratory environment, human operation, instrumentation, methodology, measurement error, etc. For example, when measuring pH, a value of approximately 5.5 should be understood to include a margin of error. As an example, when measuring formulations using an industrial pH meter, "approximately 5.5" means 5.5 ± 0.3 (i.e., pH between 5.2 and 5.8).

[0030] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments includes a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from polysorbates (e.g., polysorbate 80, polysorbate 20), poloxamer (e.g., poloxamer 188), polyhydroxyalkane, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, etc. Myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamide-propyl-betaine, myristamidopropyl-betaine, palmitoamide-propyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamide-propyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80.

[0031] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the surfactant is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of the surfactant is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of the surfactant is from 0.01 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of the surfactant is from 0.02 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 0.3 mg / mL. In some embodiments, the concentration of the surfactant is from 0.3 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the surfactant is about 0.02 mg / mL. In some embodiments, the concentration of the surfactant is about 0.2 mg / mL. In some embodiments, the concentration of the surfactant is about 0.4 mg / mL. In some embodiments, the concentration of the surfactant is about 0.6 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the surfactant concentration is 0.02 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL. In some embodiments, the surfactant concentration is 0.4 mg / mL. In some embodiments, the surfactant concentration is 0.6 mg / mL.

[0032] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of polysorbate 80 is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.01 mg / mL to 0.8 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.02 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 0.6 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 0.3 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.3 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.02 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.2 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 about 0.6 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of polysorbate 80 is 0.02 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.2 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.6 mg / mL.

[0033] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a sugar. In some embodiments, the sugar is selected from conventional compositions (CH2O). nAnd its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. In some embodiments, the sugar is selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, sylitol, sorbitol, mannitol, melitriose, maltotriose, stachyose, maltose, lactulose, maltitol, maltitol, lactitol, isomaltulose, etc. In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose.

[0034] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the sugar concentration is from 10 mg / mL to 120 mg / mL. In some embodiments, the sugar concentration is from 30 mg / mL to 100 mg / mL. In some embodiments, the sugar concentration is from 30 mg / mL to 80 mg / mL. In some embodiments, the sugar concentration is from 50 mg / mL to 100 mg / mL. In some embodiments, the sugar concentration is from 65 mg / mL to 95 mg / mL. In some embodiments, the sugar concentration is from 64 mg / mL to 96 mg / mL. In some embodiments, the sugar concentration is from 72 mg / mL to 88 mg / mL. In some embodiments, the sugar concentration is from 32 mg / mL to 48 mg / mL. In some embodiments, the sugar concentration is from 36 mg / mL to 44 mg / mL. In some embodiments, the sugar concentration is about 30 mg / mL. In some embodiments, the sugar concentration is about 40 mg / mL. In some embodiments, the sugar concentration is about 80 mg / mL. In some embodiments, the sugar concentration is about 100 mg / mL. In some embodiments, the sugar concentration is 10 mg / mL, 20 mg / mL, 30 mg / mL, 33 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 72 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the sugar concentration is 30 mg / mL. In some embodiments, the sugar concentration is 40 mg / mL. In some embodiments, the sugar concentration is 80 mg / mL. In some embodiments, the concentration of the sugar is 100 mg / mL.

[0035] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of sucrose is from 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of sucrose is from 30 mg / mL to 100 mg / mL. In some embodiments, the concentration of sucrose is from 30 mg / mL to 80 mg / mL. In some embodiments, the concentration of sucrose is from 50 mg / mL to 100 mg / mL. In some embodiments, the concentration of sucrose is from 65 mg / mL to 95 mg / mL. In some embodiments, the concentration of sucrose is from 64 mg / mL to 96 mg / mL. In some embodiments, the concentration of sucrose is from 72 mg / mL to 88 mg / mL. In some embodiments, the concentration of sucrose is from 32 mg / mL to 48 mg / mL. In some embodiments, the concentration of sucrose is from 36 mg / mL to 44 mg / mL. In some embodiments, the concentration of sucrose is about 30 mg / mL. In some embodiments, the concentration of sucrose is about 40 mg / mL. In some embodiments, the concentration of sucrose is about 80 mg / mL. In some embodiments, the concentration of sucrose is about 100 mg / mL. In some embodiments, the concentration of sucrose is 10 mg / mL, 20 mg / mL, 30 mg / mL, 33 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 72 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the concentration of sucrose is 30 mg / mL. In some embodiments, the concentration of sucrose is 40 mg / mL. In some embodiments, the concentration of sucrose is 80 mg / mL. In some embodiments, the concentration of sucrose is 100 mg / mL.

[0036] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments further comprises an excipient. In some embodiments, the excipient is ethylenediaminetetraacetic acid (EDTA) or a salt thereof, EDTA hydrate or a salt thereof, glycine, DTPA (diethylenetriaminepentaacetic acid), arginine hydrochloride, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, or calcium chloride. In some embodiments, the excipient is a sodium salt of EDTA hydrate or glycine. In some embodiments, the excipient is disodium EDTA dihydrate or glycine. In some embodiments, the excipient is a sodium salt of EDTA hydrate. In some embodiments, the excipient is disodium EDTA dihydrate. In some embodiments, the excipient is glycine.

[0037] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the excipient is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of the excipient is from 0.04 mg / mL to 0.06 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 0.05 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 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 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, or any range between these values. In some embodiments, the concentration of the excipient is 0.01 mg / mL. In some embodiments, the concentration of the excipient is 0.05 mg / mL. In some embodiments, the concentration of the excipient is 0.1 mg / mL.

[0038] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.04 mg / mL to 0.06 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.01 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.05 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.1 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 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, or any range between these values. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.01 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.05 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.1 mg / mL.

[0039] In some embodiments, in the pharmaceutical composition as described in any of the preceding embodiments, the concentration of the excipient is from 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of the excipient is from 7 mg / mL to 11 mg / mL. In some embodiments, the concentration of the excipient is from 8 mg / mL to 10 mg / mL. In some embodiments, the concentration of the excipient is about 9 mg / mL. In some embodiments, the concentration of the excipient is 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / mL, or any range between these values. In some embodiments, the concentration of the excipient is 9 mg / mL.

[0040] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of glycine is from 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of glycine is from 7 mg / mL to 11 mg / mL. In some embodiments, the concentration of glycine is from 8 mg / mL to 10 mg / mL. In some embodiments, the concentration of glycine is about 9 mg / mL. In some embodiments, the concentration of glycine is 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / mL, or any range between these values. In some embodiments, the concentration of glycine is 9 mg / mL.

[0041] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0042] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 1 mg / mL to 50 mg / mL,

[0043] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0044] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0045] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and

[0046] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0.

[0047] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0048] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0049] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL,

[0050] (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL,

[0051] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.5 mg / mL, and

[0052] (e) A 5 mM to 40 mM histidine-histidine hydrochloride buffer or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0053] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0054] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0055] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.6 mg / mL,

[0056] (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL,

[0057] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0058] (e) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0059] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0060] (a) The anti-DLL3 antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0061] (b) Polysorbate 80 at concentrations of 0.3 mg / mL to 0.5 mg / mL,

[0062] (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL,

[0063] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0064] (e) an 8 mM to 12 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0065] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0066] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0067] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL,

[0068] (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL,

[0069] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0070] (e) A 5 mM to 20 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0071] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0072] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0073] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL,

[0074] (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL,

[0075] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0076] (e) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0077] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0078] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0079] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0080] (c) Approximately 80 mg / mL of sucrose,

[0081] (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0082] (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0083] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0084] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0085] (b) 0.4 mg / mL of polysorbate 80,

[0086] (c) 80 mg / mL sucrose,

[0087] (d) 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0088] (e) 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0089] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0090] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0091] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0092] (c) Approximately 80 mg / mL of sucrose,

[0093] (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0094] (e) about 9.2 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

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

[0096] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0097] (b) 0.4 mg / mL of polysorbate 80,

[0098] (c) 80 mg / mL sucrose,

[0099] (d) 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0100] (e) A 9.2 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

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

[0102] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0103] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0104] (c) Approximately 80 mg / mL of sucrose,

[0105] (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0106] (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

[0107] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0108] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0109] (b) 0.4 mg / mL of polysorbate 80,

[0110] (c) 80 mg / mL sucrose,

[0111] (d) 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0112] (e) 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.5.

[0113] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0114] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0115] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0116] (c) Approximately 80 mg / mL of sucrose,

[0117] (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0118] (e) about 9.2 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.6.

[0119] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0120] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0121] (b) 0.4 mg / mL of polysorbate 80,

[0122] (c) 80 mg / mL sucrose,

[0123] (d) 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0124] (e) A 9.2 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.6.

[0125] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0126] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 1 mg / mL to 50 mg / mL,

[0127] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0128] (c) Sugars ranging from 10 mg / mL to 120 mg / mL, and

[0129] (d) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0.

[0130] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0131] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0132] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL,

[0133] (c) Sucrose at concentrations from 30 mg / mL to 100 mg / mL, and

[0134] (d) A 5 mM to 40 mM histidine-histidine hydrochloride buffer or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

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

[0136] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0137] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.6 mg / mL,

[0138] (c) Sucrose at concentrations from 30 mg / mL to 100 mg / mL, and

[0139] (d) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0140] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0141] (a) The anti-DLL3 antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0142] (b) Polysorbate 80 at concentrations of 0.3 mg / mL to 0.5 mg / mL,

[0143] (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL, and

[0144] (d) An 8 mM to 12 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0145] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0146] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0147] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL,

[0148] (c) Sucrose at concentrations from 30 mg / mL to 100 mg / mL, and

[0149] (d) A 5 mM to 20 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

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

[0151] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0152] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL,

[0153] (c) Sucrose at concentrations from 30 mg / mL to 100 mg / mL, and

[0154] (d) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0155] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0156] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0157] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0158] (c) Approximately 80 mg / mL of sucrose, and

[0159] (d) About 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0160] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0161] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0162] (b) 0.4 mg / mL of polysorbate 80,

[0163] (c) 80 mg / mL sucrose, and

[0164] (d) 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

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

[0166] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

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

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

[0169] (d) About 9.2 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0170] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0171] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0172] (b) 0.4 mg / mL of polysorbate 80,

[0173] (c) 80 mg / mL sucrose, and

[0174] (d) A 9.2 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0175] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0176] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0177] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0178] (c) Approximately 80 mg / mL of sucrose, and

[0179] (d) About 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

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

[0181] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

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

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

[0184] (d) 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.5.

[0185] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0186] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0187] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0188] (c) Approximately 80 mg / mL of sucrose, and

[0189] (d) About 9.2 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.6.

[0190] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0191] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0192] (b) 0.4 mg / mL of polysorbate 80,

[0193] (c) 80 mg / mL sucrose, and

[0194] (d) A 9.2 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.6.

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

[0196] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 1 mg / mL to 50 mg / mL,

[0197] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0198] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0199] (d) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and

[0200] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0.

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

[0202] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0203] (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL,

[0204] (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL,

[0205] (d) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and

[0206] (e) A 5 mM to 40 mM histidine-histidine hydrochloride buffer or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

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

[0208] (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL,

[0209] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.6 mg / mL,

[0210] (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL,

[0211] (d) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and

[0212] (e) A 5 mM to 40 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0213] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0214] (a) The anti-DLL3 antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL,

[0215] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.3 mg / mL,

[0216] (c) Sucrose at concentrations of 32 mg / mL to 48 mg / mL,

[0217] (d) Glycine at concentrations of 7 mg / mL to 11 mg / mL, and

[0218] (e) A 25 mM to 35 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0219] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0220] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0221] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0222] (c) Approximately 40 mg / mL of sucrose,

[0223] (d) Approximately 9 mg / mL of glycine, and

[0224] (e) about 30 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0225] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0226] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0227] (b) 0.2 mg / mL of polysorbate 80,

[0228] (c) 40 mg / mL sucrose,

[0229] (d) 9 mg / mL glycine, and

[0230] (e) 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

[0231] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0232] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0233] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0234] (c) Approximately 40 mg / mL of sucrose,

[0235] (d) Approximately 9 mg / mL of glycine, and

[0236] (e) about 27.8 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

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

[0238] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0239] (b) 0.2 mg / mL of polysorbate 80,

[0240] (c) 40 mg / mL sucrose,

[0241] (d) 9 mg / mL glycine, and

[0242] (e) 27.8 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8.

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

[0244] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0245] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0246] (c) Approximately 40 mg / mL of sucrose,

[0247] (d) Approximately 9 mg / mL of glycine, and

[0248] (e) about 30 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

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

[0250] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0251] (b) 0.2 mg / mL of polysorbate 80,

[0252] (c) 40 mg / mL sucrose,

[0253] (d) 9 mg / mL glycine, and

[0254] (e) 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.5.

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

[0256] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0257] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0258] (c) Approximately 40 mg / mL of sucrose,

[0259] (d) Approximately 9 mg / mL of glycine, and

[0260] (e) about 27.8 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5.

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

[0262] (a) 20 mg / mL of the aforementioned anti-DLL3 antibody-drug conjugate,

[0263] (b) 0.2 mg / mL of polysorbate 80,

[0264] (c) 40 mg / mL sucrose,

[0265] (d) 9 mg / mL glycine, and

[0266] (e) 27.8 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.5.

[0267] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, when stored at 2-8°C for 3 months, exhibits a change in SEC of no more than 0.5%, a change in R-CE of no more than 0.1%, and a change in free toxin content of no more than 4.3 ppm, or

[0268] When stored at 2-8°C for 12 months, the SEC change is no more than 1.0%, the R-CE change is no more than 0.4%, and the free toxin content change is no more than 12.5 ppm.

[0269] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0270] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0271] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0272] (c) Approximately 80 mg / mL of sucrose,

[0273] (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0274] (e) about 9.2 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.6;

[0275] or

[0276] (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL,

[0277] (b) Approximately 0.4 mg / mL of polysorbate 80,

[0278] (c) Approximately 80 mg / mL of sucrose,

[0279] (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0280] (e) about 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.5;

[0281] When the pharmaceutical composition is stored at 2-8°C for 3 months, the SEC change is no more than 0.5%, the R-CE change is no more than 0.1%, and the free toxin content change is no more than 4.3 ppm.

[0282] When stored at 2-8°C for 12 months, the SEC change is no more than 1.0%, the R-CE change is no more than 0.4%, and the free toxin content change is no more than 12.5 ppm.

[0283] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0284] This disclosure also provides a method for preparing a lyophilized formulation, including the step of lyophilizing a pharmaceutical composition as described in any of the preceding claims.

[0285] This disclosure also provides a lyophilized formulation obtained by the method described above.

[0286] This disclosure also provides a lyophilized formulation, which, upon reconstitution, can form a pharmaceutical composition as described in any of the preceding claims.

[0287] This disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted a lyophilized formulation as described in any of the preceding claims.

[0288] This disclosure also provides a reconstituted solution, which is a reconstituted form of the lyophilized formulation as described in any of the preceding claims.

[0289] In some embodiments, the reconstituted solution as described in any of the preceding claims has the same components and contents as the pharmaceutical composition as described in any of the preceding claims.

[0290] This disclosure also provides an article of manufacture comprising a container containing a pharmaceutical composition as described in any of the preceding claims, or a lyophilized formulation as described in any of the preceding claims.

[0291] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is an intravenous injection formulation.

[0292] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is suitable for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the preceding embodiments is suitable for intravenous injection.

[0293] In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is used to prepare a drug for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is used to prepare a drug for intravenous injection.

[0294] This disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described in any of the preceding claims, a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0295] This disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described in any of the preceding claims, or a lyophilized formulation as described in any of the preceding claims.

[0296] This disclosure also provides the use of pharmaceutical compositions as described in any of the preceding claims, or lyophilized formulations as described in any of the preceding claims, in the preparation of medicaments for the treatment or prevention of diseases.

[0297] This disclosure also provides pharmaceutical compositions as described in any of the preceding claims, or lyophilized formulations as described in any of the preceding claims, for use as medicines. In some embodiments, the medicine is used to treat or prevent disease.

[0298] In some implementations, the disease is a tumor or cancer.

[0299] In some implementations, the disease is a solid tumor.

[0300] In some implementations, the diseases are selected from: lung cancer (e.g., small cell lung cancer, large cell lung cancer), melanoma, medullary thyroid carcinoma, glioblastoma, neuroendocrine tumors, prostate cancer, glioma, thyroid cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), brain cancer, glioblastoma multiforme, neuroblastoma, central nervous system cancer, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer (e.g., stomach cancer, intestinal cancer, colorectal cancer), kidney cancer (e.g., clear cell renal cell carcinoma), ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, testicular cancer, adrenal cancer, and skin cancer.

[0301] In some implementations, the diseases are selected from: lung cancer, melanoma, medullary thyroid carcinoma, glioblastoma, neuroendocrine tumors, prostate cancer, glioma, thyroid cancer, head and neck cancer, brain cancer, glioblastoma multiforme, neuroblastoma, central nervous system cancer, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, malignant pleural mesothelioma, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, testicular cancer, adrenal cancer, and skin cancer.

[0302] In some implementations, the disease is small cell lung cancer. Attached Figure Description

[0303] Figures 1A to 1C show the binding of Hu6 and Hu100 antibodies to DLL3 from different species. Specifically: Figure 1A shows the FACS results of Hu6 and Hu100 antibodies binding to H1184 cells; Figure 1B shows the FACS results of Hu6 and Hu100 antibodies binding to cynoDLL3 / CHO-s cells; Figure 1C shows the FACS results of Hu6 and Hu100 antibodies binding to ratDLL3 / CHO-s cells.

[0304] Figure 2 shows the results of competitive binding experiments with different anti-DLL3 antibodies. The results show that Hu6 and Hu100 do not compete with BI-764532, indicating that antibodies Hu6 and Hu100 bind to different epitopes of BI-764532.

[0305] Figure 3 shows the results of endocytosis of Hu6 and Hu100 antibodies within cells. The results indicate that both Hu6 and Hu100 can be endocytosed.

[0306] Figures 4A and 4B show the binding results of ADC-1, ADC-2, and ADC-3 on cells expressing human or monkey DLL3. Specifically: Figure 4A shows the binding results of ADC-1, ADC-2, and ADC-3 on cells expressing human DLL3; Figure 4B shows the binding results of ADC-1, ADC-2, and ADC-3 on cells expressing monkey DLL3.

[0307] Figures 5A to 5D show the growth-inhibiting effects of ADC-1, ADC-2, and ADC-3 on different cell types. Specifically: Figure 5A shows the growth-inhibiting effects of ADC-1, ADC-2, and ADC-3 on H1184 cells highly expressing DLL3; Figure 5B shows the growth-inhibiting effects of ADC-1, ADC-2, and ADC-3 on DMS53 cells highly expressing DLL3; Figure 5C shows the growth-inhibiting effects of ADC-1, ADC-2, and ADC-3 on SK-MEL3 cells with low DLL3 expression; and Figure 5D shows the growth-inhibiting effects of ADC-1, ADC-2, and ADC-3 on CHO-K1 cells that do not express DLL3.

[0308] Figure 6 shows the bystander cytotoxicity results for ADC-1, ADC-2, and ADC-3.

[0309] Figure 7 shows the results of ADC-1, ADC-2, ADC-4, and ADC-5 inhibiting the growth of subcutaneously transplanted DMS53 cells in mice.

[0310] Figure 8 shows the results of ADC-1 and ADC-3 inhibiting the growth of subcutaneously transplanted DMS53 cells in mice.

[0311] Figure 9 shows the results of ADC-2, ADC-3, ADC-6, and ADC-7 inhibiting the growth of subcutaneously transplanted H1184 cell tumors in mice.

[0312] Figures 10A to 10C show the pharmacokinetic results of ADC-1, ADC-2, and ADC-3 rats. Specifically: Figure 10A shows the pharmacokinetic results of ADC-1 rats; Figure 10B shows the pharmacokinetic results of ADC-2 rats; and Figure 10C shows the pharmacokinetic results of ADC-3 rats.

[0313] Figure 11 shows the pharmacokinetic results of ADC-2 in cynomolgus monkeys. Detailed Implementation

[0314] the term

[0315] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0316] The singular forms “a,” “an,” and “the” used in this disclosure include plural references unless the context clearly indicates otherwise.

[0317] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0318] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0319] Those skilled in the art will understand that when used as a reference range, cutoff value, or specific value, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range with a difference of up to 20% (i.e., ±20%). Since many of the values ​​used herein were determined experimentally, those skilled in the art will understand that such determinations can vary between different experiments and are generally true across experiments. Due to this inherent variability, the values ​​used herein should not be unduly restrictive. Therefore, the term "about" is used to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% or less from a specified value.

[0320] Although this disclosure provides content ranges or content values, those skilled in the art will understand that the content ranges or content values ​​cover the acceptable range of error for the specific values ​​measured.

[0321] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0322] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics 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 subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0323] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetraglycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to C terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The IgG heavy chain constant region (CH) typically contains three constant domains (CH1, CH2, and CH3); similarly, from the N to C terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0324] The term "variable region" or "variable domain" refers to the domain in the antibody heavy or light chain involved in antibody-antigen binding. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0325] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0326] Table 1. Relationship between CDR numbering systems

[0327] Unless otherwise stated, the variable regions and CDR sequences in this disclosure embodiment are subject to the "Kabat" numbering rule.

[0328] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0329] The antibodies disclosed herein may be derived from animals (such as antibodies from mice, birds, rabbits, camels, monkeys, etc.), chimeric antibodies, or humanized antibodies.

[0330] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0331] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0332] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art.

[0333] The terms "anti-DLL3 antibody" and "DLL3-binding antibody" refer to antibodies capable of binding to DLL3 or its epitopes with sufficient affinity. In one embodiment, the anti-DLL3 antibody binds to unrelated proteins to a degree less than at least about 10% of the antibody's binding to DLL3, and this binding can be measured by BIACORE surface plasmon resonance assay.

[0334] The term "antigen" refers to a molecule or molecular moiety that can be bound by a selective binder of an antigen-binding protein (such as an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (such as antibodies).

[0335] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen as a protein), which allows non-consecutive amino acids to be spatially close. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

[0336] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 An equilibrium dissociation constant (KD) of M or less binds to an antigen or epitope. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset).

[0337] Antibody drug conjugates (ADCs) are conjugates obtained by linking an antibody (or its antigen-binding fragment) directly or through a linker to a drug.

[0338] A "drug" (abbreviated as D) is any substance that has biological or detectable activity (e.g., therapeutic agents, detectable markers, binders, etc.) and its prodrugs, which are metabolized in the body to become active agents. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors, and immunomodulators. Chemotherapeutic agents are chemical compounds that can be used to treat cancer. Representative therapeutic agents include cytotoxins, cytotoxic agents, and cell growth inhibitors.

[0339] Cytotoxicity refers to the loss, elimination, and / or killing of target cells. Cytotoxic agents are drugs that have cytotoxic and / or cell growth-inhibiting effects on cells. Cell growth inhibition refers to the inhibition of cell proliferation. Cell growth inhibitors are drugs that have a cell growth-inhibiting effect on cells, thereby inhibiting the growth and / or expansion of specific subgroups of cells.

[0340] Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, apoptosis-promoting agents, and cell-lysing enzymes (e.g., RNase). These drug descriptive terms are not mutually exclusive, and therefore, one or more of the aforementioned terms may be used to describe a therapeutic agent. For example, the selected radioisotope may also be a cytotoxic agent. Therapeutic agents can be prepared as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Generally, conjugates containing a radioisotope as a drug are called radioimmunoconjugates, and those containing a chemotherapeutic agent as a drug are called chemoimmunoconjugates.

[0341] Examples of cytotoxic agents include, but are not limited to, anthracycline, orrisstatin, CC-1065, dolastatin, docalmicin, enediyne, geldanamycin, maytansine, puromycin, taxane, vinca alkaloids, SN-38, tubulolysin, hemiasterlin, eribulin, trabectedin, lurbinectedin, and their stereoisomers, isosteres, analogues, or derivatives. Chemotherapy agents, phytotoxicants, other bioactive proteins, enzymes (i.e., ADEPT), radioactive isotopes, and photosensitizers (i.e., for photodynamic therapy) may also be used.

[0342] The terms "connector unit" and "connector" refer to a chemical structural fragment or bond that is linked to an antibody at one end and a drug at the other. Connectors can also be attached to other connectors before being linked to an antibody or drug. Connector attachment to antibodies can be accomplished in various ways, such as via surface lysine residues, reductive coupling to oxidized carbohydrates, release of cysteine ​​residues via reducing interchain disulfide bonds, modification of reactive cysteine ​​residues at specific sites, and tags containing acyl donor glutamine, or modification of peptides to make them reactive endogenous glutamine in the presence of transglutaminase and amines. Various ADC linker systems are known in the art, including hydrazone-, disulfide-, and peptide-based links.

[0343] The connector may comprise one or more connector elements. Exemplary connector elements include 6-maleiminohexanoyl (“MC”), maleiminopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimino-4-(2-pyridylthio)valerate (“SPP”), N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (“SMCC”, also referred to herein as “MCC”), and N-succinimino-(4-iodo-acetyl)aminobenzoate (“SIAB”).

[0344] The linker may be selected from the following elements or combinations thereof: extensions, spacers, and amino acid units. The linker may be synthesized by methods known in the art, such as those described in US20050238649A1. The linker may be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photostable linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

[0345] “LD” is the connector-drug section formed when the drug (D) is connected to the connector (L).

[0346] "Drug loading," also known as drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated to each antibody in an ADC. It can range from about 1 to about 10 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 1-3, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. The general formula of the ADC disclosed herein includes a set of antibody-drug conjugates within the aforementioned range. In embodiments disclosed herein, drug loading may be expressed as n, which can be a decimal or an integer. Drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, HIC, and RP-HPLC.

[0347] "Surfactant" refers to a surface-active agent, preferably a nonionic surfactant. Surfactants can reduce protein aggregation and / or particle formation in formulations. The amount of surfactant added is such that it reduces protein aggregation and minimizes particle formation in the formulation.

[0348] "Poloxamer" is an α-hydro-ω-hydroxy poly(oxyethylene) a -Poly(oxypropylene) b - Poly(ethylene oxide) a Block copolymers. These are formed by reacting propylene oxide and propylene glycol to form polyoxypropylene glycol, followed by the addition of ethylene oxide to form a block copolymer. Here, "a" represents the number of ethylene oxide units, and "b" represents the number of propylene oxide units. Examples of poloxamers include poloxamer 188. Specifically, poloxamer 188 has 75–85 ethylene oxide units (a) and 25–30 propylene oxide units (b) in the copolymer, with an ethylene oxide (EO) content of 79.9%–83.7% and an average molecular weight of 7680–9510.

[0349] In this disclosure, "disodium edetate", "disodium ethylenediaminetetraacetate dihydrate" and "Na2EDTA·2H2O" can be used interchangeably, and they are all substances with CAS number: 6381-92-6.

[0350] "Viscosity modifier" is a conventional pharmaceutical excipient added to adjust the viscosity of a formulation. Viscosity modifiers can be inorganic salts and amino acid salts. Preferably, the inorganic salt is selected from sodium chloride, calcium chloride, and magnesium chloride. More preferably, the amino acid salt is selected from arginine hydrochloride, histidine hydrochloride, lysine hydrochloride, histidine acetate, etc.

[0351] "Buffer" refers to a buffering agent that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate (also known as tartrate), tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0352] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine acetate, histidine-histidine hydrochloride, histidine-histidine phosphate, and histidine-histidine sulfate buffers, with histidine-histidine acetate buffers being preferred. Histidine-histidine acetate buffers can be prepared by reacting histidine with acetic acid, or by reacting histidine with histidine acetate.

[0353] "Displacement" refers to the replacement of the solvent system in which the protein is dissolved. For example, a buffer system used in a stabilizing formulation may be physically replaced by a high-salt or hypertonic solvent system containing the protein, thereby allowing the protein to remain in the stabilizing formulation. These physical methods include, but are not limited to, ultrafiltration, dialysis, or centrifugation.

[0354] "Pharmaceutical composition" means a mixture containing one or more antibody-drug conjugates or their physiologically / pharmacologically acceptable salts or prodrugs described herein, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0355] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to the salts of the antibody-drug conjugates disclosed herein, which are safe and effective when used in subjects and possess the intended biological activity. As an example, the antibody-drug conjugates disclosed herein contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0356] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and R. Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0357] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by vacuum freeze-drying a liquid or solution preparation. Typically, freeze-drying includes pre-freezing, primary drying, and secondary drying. Pre-freezing aims to freeze the product to obtain a crystalline solid; in some embodiments, the pre-freezing temperature is set to -45°C, and the pre-freezing rate is set to 1°C / min. Primary drying, also known as main drying, is the main stage of sample freeze-drying and aims to remove ice from the product while maintaining its shape and minimizing damage. Improper selection of the temperature and vacuum level during primary drying can lead to product collapse; higher temperatures and vacuum levels increase freeze-drying efficiency but also increase the risk of product collapse. In some embodiments, the primary drying temperature can be a temperature conventional in the art, such as -30°C to 0°C. Secondary drying, also known as desorption drying, is the main step of removing bound water from the product by applying an ultimate vacuum (0.01 mbar) and increasing the temperature (20°C to 40°C). Because most biological products are temperature-sensitive, the secondary drying temperature is often chosen at the lower end of the temperature range, such as 25°C. The freeze-drying time depends on the freezer, the dosage of the freeze-dried formulation, and the container of the freeze-dried drug. Such adjustments are well known to those skilled in the art.

[0358] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0359] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0360] The pharmaceutical compositions disclosed herein achieve a stable effect: the antibody-drug conjugates therein substantially retain their physical and / or chemical stability and / or biological activity after storage; preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0361] Stable formulations include those in which no significant changes are observed when stored at refrigerated temperatures (2°C–8°C) for at least 1 month, at least 3 months, at least 6 months, preferably 1 year, and even more preferably up to 2 years. Stable liquid formulations also include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, or 6 months. Furthermore, stable liquid formulations also include those that exhibit the desired characteristics after storage at 40°C for periods of 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include antibody-drug conjugates showing aggregation or degradation typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly milky white. The concentration, pH, weight, and molecular osmotic pressure of the formulation exhibit aggregation of not more than about 10%, preferably not more than about 5%.

[0362] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody-drug conjugate does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody-drug conjugate "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0363] If an antibody-drug conjugate does not exhibit significant chemical changes, then the antibody-drug conjugate "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0364] If the biological activity of an antibody-drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the drug formulation is prepared, then the antibody-drug conjugate "retains its biological activity" in the drug formulation.

[0365] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0366] "Treatment" means administering an oral or topical therapeutic agent, such as a pharmaceutical composition comprising any of the substances disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, in order to induce the regression of such symptoms or inhibit their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0367] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate a diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects disclosed herein may be animal or human subjects.

[0368] The pharmaceutical compositions disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion. In some embodiments, the pharmaceutical compositions disclosed herein are administered by intravenous injection.

[0369] The pharmaceutical compositions disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. Optionally, the pharmaceutical composition may also be formulated with one or more other agents for the prevention or treatment of said condition. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the pharmaceutical composition, the type of condition or treatment, and other factors. It may be used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and any route of administration determined empirically / clinically to be appropriate.

[0370] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0371] Example

[0372] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0373] Experimental methods not specifying specific conditions in the examples or test cases disclosed herein are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, standard reagents.

[0374] I. Antibody Preparation

[0375] The anti-DLL3 antibody disclosed herein is derived from CN202310172129.X, CN202311485274.X, and PCT / CN2024 / 078818 (included herein in their entirety by reference).

[0376] Example 1: Preparation of DLL3 antigen, detection protein, and stable cell line

[0377] DLL3 genes from different species, along with human DLL1 and DLL4 genes, were transfected into Chinese hamster ovary CHO-s cells (Invitrogen, R80007) to construct CHO-s cell lines expressing DLL3 protein from different species. These cells were then used for subsequent antibody screening and identification. The amino acid sequences of the relevant proteins are as follows:

[0378] Human DLL3 full-length protein (Uniprot, Q9NYJ7):

[0379] Cynomolgus monkey DLL3 full-length protein (Uniprot, A0A2K5WSR4):

[0380] Rat DLL3 full-length protein (Uniprot, O88671):

[0381] Mouse DLL3 full-length protein (Uniprot, O88516):

[0382] Human DLL1 full-length protein (Uniprot, O00548):

[0383] Human DLL4 full-length protein (Uniprot, Q9NR61):

[0384] 1.1 Construction of cell lines expressing high levels of DLL3, DLL1, and DLL4

[0385] The pCDH lentiviral expression vector plasmids (synthesized by GENEWIZ) containing SEQ ID NO: 1-6 were transfected into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent, along with pVSVG and pCMV lentiviral packaging vectors. The virus-containing culture supernatant was collected, filtered, and ultracentrifuged. After discarding the supernatant, the cells were resuspended in 0.2 mL of sterile PBS. The concentrated virus was used to infect Chinese hamster ovary cells CHO-s (Invitrogen, R80007), DMS53 (ATCC, CRL-2062), and H82 (ATCC, HTB-175), respectively. After selection with puromycin for two to three weeks, single-cell sorting was performed using FACS. The selected monoclonal cell lines were expanded and cryopreserved.

[0386] 1.2 Preparation of Antigen

[0387] Using human DLL3 (Uniprot, Q9NYJ7), cynomolgus monkey DLL3 (Uniprot, A0A2K5WSR4), and mouse DLL3 (Uniprot, O88516) sequences as templates, DLL3 ECD fusion proteins with different tags were designed, cloned into the pTT5 vector, and expressed in 293E cells to obtain the antigens. The relevant protein amino acid sequences are as follows:

[0388] 1) His-hDLL3(ECD):

[0389] Note: The dotted and dashed lines represent partial signal peptide sequences. The single-strike part represents the his tag and linker, and the double-strike part represents the extracellular region of DLL3.

[0390] 2) Fc-hDLL3(ECD):

[0391] Note: The dotted and dashed portions represent the signal peptide sequence, the single-strike portion represents the Fc tag and linker, and the double-strike portion represents the DLL3 extracellular region.

[0392] 3) hDLL3(ECD)-strep twin:

[0393] Note: The dotted and dashed parts are signal peptide sequences, the double-dashed parts are the extracellular region of DLL3, and the single-dashed parts are strep twin tags.

[0394] 4) cynoDLL3(ECD)-strep twin:

[0395] Note: The dotted line part is the signal peptide sequence, the double dotted line part is the extracellular region of DLL3, and the single dotted line part is the strep twin tag.

[0396] 5) mouDLL3(ECD)-strep twin:

[0397] Note: The dotted line part is the signal peptide sequence, the double dotted line part is the extracellular region of DLL3, and the single dotted line part is the strep twin tag.

[0398] Example 2: Preparation of Mouse Anti-Human DLL3 Monoclonal Antibody

[0399] 1. Immunization

[0400] The mouse anti-human DLL3 monoclonal antibody was generated by immunizing mice. SJL mice, female, 6 - 8 weeks old (Shanghai Slack Experimental Animal Co., Ltd., Animal Production License No.: SCXK(Shanghai)2017 - 0005) were used in the experiment. Feeding environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle adjustment, temperature 20 - 25 °C; humidity 40 - 60%. The mice that had adapted to the environment were immunized according to the following protocol.

[0401] Immunization protocol:

[0402] The immunization antigen for the first group of mice was His-hDLL3(ECD) (SEQ ID NO: 7). Use Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No.77161) adjuvants for cross-immunization. The ratio of antigen to Gold Adjuvant was 1:1, and the ratio of antigen to Thermo Alum was 3:1, 50 μg / mouse / time (primary immunization), 25 μg / mouse / time (boost immunization). After the antigen was emulsified, inoculation was carried out, and the immunization times were 0, 7, 14, and 21 days. Blood was taken on the 7th and 21st days, and the antibody titer in the mouse serum was determined by the ELISA method. After the 4th immunization, mice with high antibody titers in the serum and a titer tending to plateau were selected for splenocyte fusion. Three days before splenocyte fusion, boost immunization was carried out by intraperitoneal (i.p.) injection of an antigen solution prepared with 25 μg / mouse of physiological saline.

[0403] The second group of mice was immunized with DLL3 CHO-s and Fc-hDLL3(ECD) (SEQ ID NO: 8) antigens, and the immunization method was alternating immunization with cell and protein antigens. Before the first immunization of DLL3 CHO-s cells, [they were] pre-treated with [a specific treatment]. Gold Adjuvant (Sigma Cat No. T2684) 0.1 mL / mouse was injected intraperitoneally. Half an hour later, 0.1 mL of the solution diluted with physiological saline was injected intraperitoneally into each mouse. 8 Cell slurry concentration / mL. After evenly dispersing the cells, inoculate them on days 0, 14, 28, and 42. 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. The Gold Adjuvant ratio is 1:1, with the antigen and adjuvant Thermo... The alum ratio was 3:1, with 50 μg / mouse / dose (initial immunization) and 25 μg / mouse / dose (booster immunization). hDLL3-Fc immunization was performed on days 7, 21, 35, and 49. Blood samples were collected on days 14, 35, and 49, and serum antibody titers were determined using ELISA. After the 8th immunization, mice with high serum antibody titers that were trending towards a plateau were selected for spleen cell fusion. A booster immunization was performed 3 days prior to spleen cell fusion, with an intraperitoneal (ip) injection of 50 μg / mouse of hDLL3-Fc protein antigen solution prepared with physiological saline.

[0404] 2. Spleen cell fusion

[0405] An optimized electrofusion method was used to fuse splenic lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0406] Fusion hybridoma cells at 3-4 × 10 5 The culture medium was resuspended at a density of 1 / mL in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI), and 150 μL / well was seeded into 96-well plates. After incubation at 37°C and 5% CO2 for 3-4 days, the supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. After incubation at 37°C and 5% CO2 for 3 days, screening and detection were performed.

[0407] 3. Hybridoma cell screening and antibody sequencing

[0408] Based on the hybridoma cell growth density, the hybridoma culture supernatant was analyzed using ELISA (for DLL3 protein binding) and FACS (for DLL3 CHO-s cells binding). Clones binding human DLL3 protein, monkey DLL3 protein, and DLL3 CHO-s cells, while not binding wild-type CHO-s cells, were selected and promptly cryopreserved, expanded, preserved, and subjected to one to two subcloning processes until single-cell clones were obtained. Hybridoma clones mAb100 and mAb6 were obtained through the above experiments.

[0409] The hybridoma clone was cultured and RNA was extracted. Reverse transcription amplification (RT-PCR) was performed using degenerate primers for mouse-Ig to obtain the variable region sequence of the antibody.

[0410] mAb6 heavy chain variable region:

[0411] mAb6 light chain variable region:

[0412] mAb100 heavy chain variable region:

[0413] mAb100 light chain variable region:

[0414] Table 2. CDR sequences of murine antibodies

[0415] Note: The amino acid residues of the CDRs of VH / VL are determined and annotated using the Kabat numbering system.

[0416] The heavy chain variable region and light chain variable region of the mouse antibody were cloned into the pTT 5 vector plasmid containing the human IgG1 heavy chain constant region shown in SEQ ID NO:28 and the κ light chain constant region shown in SEQ ID NO:29, respectively. Then, HEK293 cells were transfected to obtain the chimeric antibodies M6CHI and M100CHI against DLL3.

[0417] Human IgG1 heavy chain constant region:

[0418] Human κ light chain constant region:

[0419] Example 3: Humanization of mouse anti-DLL3 monoclonal antibody

[0420] By comparing the Kabat human antibody heavy and light chain variable region germline gene database, germline genes with high homology in the heavy and light chain variable regions were selected as templates. The CDRs of the murine antibody were grafted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Reversion mutations were then performed on the amino acids in the variable regions, followed by recombination with constant regions (exemplarily, with the human IgG1 heavy chain constant region shown in SEQ ID NO: 28 and the human κ light chain constant region shown in SEQ ID NO: 29) to obtain the full-length antibody.

[0421] For the human lineage of mAb6 antibody, the templates for the variable regions FR1, FR2, and FR3 of the light chain are IGKV1-16*01, and the template for the FR4 region of the light chain is IGKJ4*01. For the human lineage of mAb6 antibody, the templates for the variable regions FR1, FR2, and FR3 of the heavy chain are IGHV1-3*01, IGHV7-4-1*02, or IGHV3-73*01, and the template for the FR4 region of the heavy chain is IGHJ6*01. For the human lineage of mAb100 antibody, the templates for the variable regions FR1, FR2, and FR3 of the light chain are IGKV1-27*01, and the template for the FR4 region of the light chain is IGKJ4*01. For the human lineage of mAb100 antibody, the templates for the variable regions FR1, FR2, and FR3 of the heavy chain are IGHV3-11*01, and the template for the FR4 region of the heavy chain is IGHJ6*01.

[0422] In addition, the fourth amino acid residue in the mAb100 heavy chain variable region HCDR3:PLYYYGRSYNAVAY (SEQ ID NO: 24) was mutated from Y to H and the eleventh residue was mutated from A to G to obtain new HCDR3:PLYHYGRSYNAVAY (SEQ ID NO: 30) and PLYYYGRSYNGVAY (SEQ ID NO: 31).

[0423] Table 3. Humanized templates and corresponding point mutations for mAb6 and mAb100 antibodies

[0424] Note: Taking A100EG in the table as an example, it means that the A at position 100E under the kabat number is mutated to G.

[0425] The sequence of the variable region of the obtained humanized antibody is as follows:

[0426] hAb6 VH1(Q1E,R71V,T73K)

[0427] hAb6 VH2(Q1E,I69L,R71V,T73K,S76N)

[0428] hAb6 VH3(Q1E,M48I,V67A,I69L,R71V,T73K,S76N)

[0429] hAb6 VH4(Q1E,Q43K,I69L,R71V,T73K,S76N)

[0430] hAb6 VH5(Q1E,L71V,T73K,F69L)

[0431] hAb6 VH6(Q1E,F69L,L71V,T73K,V75S,S76N)

[0432] hAb6 VH7(F27Y,S30T,I69L,R71V,D73K,T93A)

[0433] hAb6VH8(Graft IGHV1-3*01)

[0434] hAb6VH9(Graft IGHV7-4-1*02)

[0435] hAb6VH10(Graft IGHV3-73*01)

[0436] hAb6VH11(Q1E,R38K,F69L,L71V,T73K,V75S,S76N)

[0437] hAb6VH12(Q1E,V68A,F69L,L71V,T73K,V75S,S76N)

[0438] hAb6VH13(Q1E,R38K,V68A,F69L,L71V,T73K,V75S,S76N)

[0439] hAb6 VL1(F36L,S46G)

[0440] hAb6 VL2(F36L,S46G,T69A,F71Y)

[0441] hAb6 VL3(F36L,A43S,P44F,S46G,T69A,F71Y)

[0442] hAb6 VL4(F36L,S46G,T69A,F71Y,T85D)

[0443] hAb6VL5(Graft IGKV1-16*01)

[0444] hAb100 VH1(Q1E,R94S)

[0445] hAb100 VH2(Q1E,S49A,R94S)

[0446] hAb100 VH3(Q1E,S49A,R94S,Y98H)

[0447] hAb100 VH4(Q1E,S49A,R94S,A100EG)

[0448] hAb100VH5(Graft IGHV3-11*01)

[0449] hAb100 VL1 (Graft IGKV1-27*01)

[0450] hAb100 VL2(V43I)

[0451] Note: Single-lined areas represent CDR regions, while double-lined areas represent mutation sites.

[0452] Table 4. General formula of the CDR region of hAb100 antibody

[0453] Where X1 is Y or H; X2 is A or G.

[0454] An example combination of the heavy and light chain variable regions of a humanized antibody is as follows:

[0455] Table 5. Humanized antibodies against mAb6

[0456] Note: hAb6L1H1 indicates that the antibody contains the heavy chain variable region hAb6VH1 and the light chain variable region hAb6VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 28, the sequence of its light chain constant region is SEQ ID NO: 29, and so on.

[0457] Table 6. Humanized antibodies of mAb100

[0458] Note: hAb100L1H1 indicates that the antibody contains the heavy chain variable region hAb100VH1 and the light chain variable region hAb100VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 28, the sequence of its light chain constant region is SEQ ID NO: 29, and so on.

[0459] The above antibodies were cloned, expressed, and purified separately. Through protein binding assays (Test Example 1), cell binding assays (Test Example 2), and Biacore assays (Test Example 4), the humanized antibodies with better activity were finally selected. The heavy and light chain amino acid sequences of exemplary humanized antibodies are as follows:

[0460] Hu6 (also known as hAb6L4H12) heavy chain:

[0461] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0462] SEQ ID NO: 58

[0463] Hu6 (also known as hAb6L4H12) light chain:

[0464] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0465] SEQ ID NO: 59

[0466] Hu100 (also known as hAb100L1H1) heavy chain:

[0467] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0468] SEQ ID NO: 60

[0469] Hu100 (also known as hAb100L1H1) light chain:

[0470] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0471] SEQ ID NO: 61

[0472] The positive control antibody used in this disclosure is BI-764532 (constructed according to WO2019234220A1), and the negative control is C25 (where the VH / VL sequence is from patent US6114143A), and their sequences are as follows: BI-764532 heavy chain:

[0473] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0474] SEQ ID NO: 62

[0475] BI-764532 Light Chain:

[0476] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0477] SEQ ID NO: 63

[0478] C25 heavy chain:

[0479] C25 light chain:

[0480] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0481] II. Preparation of ADC

[0482] The ADC disclosed herein is derived from CN202310172129.X, CN202311485274.X, and PCT / CN2024 / 078818 (included in this document by reference in their entirety).

[0483] Example 4. Preparation of ADC-1

[0484] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 50.7 μL, 507 nmol) was added to the PBS buffered aqueous solution of antibody Hu100 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 3 mL, 203 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0485] Compound 9-A (2.18 mg, 2030 nmol, prepared according to Example 9 of patent application WO2020063676A1) was dissolved in 150 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffered solution of the title product ADC-1 (1.61 mg / mL, 15.1 mL), which was stored frozen at 4 °C.

[0486] RP-HPLC calculated average: n = 4.43.

[0487] Example 5. Preparation of ADC-2

[0488] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 121.7 μL, 1217 nmol) was added to the PBS buffered aqueous solution of antibody Hu100 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 3 mL, 203 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0489] Compound 9-A (3.27 mg, 3042 nmol) was dissolved in 150 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.42 mg / mL, 15.8 mL) of the title product ADC-2, which was stored frozen at 4 °C.

[0490] RP-HPLC calculated average: n = 7.32.

[0491] Example 6. Preparation of ADC-3

[0492] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 159.4 μL, 1594 nmol) was added to the PBS buffered aqueous solution of antibody Hu100 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 6.5 mL, 440 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0493] Compound 9-A (5.66 mg, 5265 nmol) was dissolved in 330 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.72 mg / mL, 19.2 mL) of the title product ADC-3, which was stored frozen at 4 °C.

[0494] RP-HPLC calculated average: n = 6.12.

[0495] Example 7. Preparation of ADC-4

[0496] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 1.14 mL, 11.4 μmol) was added to the PBS buffered aqueous solution of antibody Hu6 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 76 mL, 5140 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0497] Compound 9-A (49.0 mg, 45.6 μmol) was dissolved in 3.8 mL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (4.07 mg / mL, 167 mL) of the title product ADC-4, which was stored frozen at 4 °C.

[0498] RP-HPLC calculated average: n = 3.53.

[0499] Example 8. Preparation of ADC-5

[0500] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 2.74 mL, 27.4 μmol) was added to the PBS buffered aqueous solution of antibody Hu6 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 68 mL, 4595 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0501] Compound 9-A (82.75 mg, 77.04 μmol) was dissolved in 3.4 mL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified by Sephadex G25 gel column chromatography (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer solution of the title product ADC-5 (3.12 mg / mL, 174.1 mL), which was then stored frozen at 4 °C.

[0502] RP-HPLC calculated average: n = 7.43.

[0503] Example 9. Preparation of ADC-6

[0504] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 35.6 μL, 356 nmol) was added to the PBS buffered aqueous solution of antibody C25 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.6 mL, 108 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0505] Compound 9-A (1.39 mg, 1.294 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.06 mg / mL, 12.3 mL) of the title product ADC-6, which was stored frozen at 4 °C.

[0506] RP-HPLC calculated average: n = 6.76.

[0507] Example 10. Preparation of ADC-7

[0508] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 64.8 μL, 648 nmol) was added to the PBS buffered aqueous solution of antibody C25 (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.6 mL, 108 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0509] Compound 9-A (1.74 mg, 1.62 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.03 mg / mL, 11.9 mL) of the title product ADC-7, which was stored frozen at 4 °C.

[0510] RP-HPLC calculated average: n = 7.75.

[0511] Drug loading analysis of ADC stock solution

[0512] An antibody-drug conjugate (ADC) is a drug-drug conjugate whose mechanism of action relies on the targeted delivery of drugs to cells, thereby killing or inhibiting cell growth. The amount of drug loaded plays a decisive role in the efficacy of the drug.

[0513] This disclosure uses the RP-HPLC method to analyze drug loading.

[0514] Test Example 1: ELISA assay for detecting antibody protein binding levels

[0515] Spread streptavidin (abcam, ab136200, 1 μg / mL) onto the plate at 100 μL / well and incubate overnight at 4°C. Wash the plate three times with 250 μL / well of PBST solution (PBS containing 0.1% Tween 20). Block with 5% milk at 250 μL / well and incubate 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 antibodies Hu6 and Hu100 (maximum concentration 100 nM, serially diluted 4-fold) and incubate at 37°C for 1 hour. Wash the plate six times with 250 μL / well of PBST solution. Add 100 μL of working concentration human IgG(H+L)-HRP (Jackson, 109-035-003, 1:4000 dilution) antibody to each well and incubate at 37°C for 1 hour. Wash the plate 6 times with 250 μL of PBST solution per well. Add 100 μL of TMB (KPL, 5120-0077) chromogenic buffer to each well and incubate at room temperature for 5-10 minutes. Add 100 μL of 1M H2SO4 to stop the incubation process and read the value at 450 nm using a microplate reader (Molecular Devices, VERSA max).

[0516] Table 8. Antibody binding activity to proteins

[0517] The results showed that both antibodies Hu6 and Hu100 had excellent binding ability to DLL3.

[0518] Test Example 2: Binding assay for detecting antibody levels in cells using FACS

[0519] Small cell lung cancer cell lines expressing DLL3, including H1184 (ATCC, catalog number CRL-5858), DLL3 / H82, cynoDLL3 / CHO-s, and RatDLL3 / CHO-s, were prepared into 1×10⁻⁶ cells using FACS buffer (1% BSA + pH 7.4 PBS). 6100 μL / well of cell suspension was added to each well of a 96-well round-bottom plate (Corning, 3795). The plates were centrifuged at 300 g for 5 minutes, and the supernatant was removed. Different concentrations of the test antibody were added, 100 μL / well. The plates were incubated at 4°C in the dark for 1 hour. After washing three times by centrifugation at 300 g, either APC anti-human IgG Fc (BioLegend, 410712) or PE F(ab')2-goat anti-human IgG (Invitrogen, H10104) at the working concentration were added, and the plates were incubated at 4°C in the dark for 40 minutes. After washing three times by centrifugation at 300 g, the geometric mean fluorescence intensity was detected using an Invitrogen flow cytometer, and the EC50 value of antibody binding to DLL3-expressing cells was calculated. The results are shown in Tables 9-1, 9-2, 9-3, and Figures 1A to 1C.

[0520] Table 9-1. Binding activity of antibodies to DLL3 expressed by cells

[0521] Table 9-2. Binding activity of antibodies to DLL3 / H82 cells expressing human DLL3

[0522] Table 9-3. Binding activity of antibodies to H1184 cells

[0523] The results showed that all the antibodies disclosed in this study could specifically bind to DLL3 expressed by cells. Hu6 exhibited high binding ability to cells expressing DLL3 from different species. Hu100 showed excellent binding ability to cells expressing DLL3 from both humans and cynomolgus monkeys, but did not bind to cells expressing DLL3 from rats. BI-764532 only bound to cells expressing DLL3 from both humans and cynomolgus monkeys, and its binding activity was weaker than that of Hu6 and Hu100.

[0524] Test Example 3: FACS Detection of Antibody Binding to DLL1 and DLL4 Cells

[0525] Stable human DLL1 / CHO-s and human DLL4 / CHO-s cells were prepared into 1×10⁻⁶ cells using FACS buffer (containing 1% BSA and pH 7.4 PBS). 6100 μL / well of cell suspension was added to a 96-well round-bottom plate (Corning, 3795). The plate was centrifuged at 300 g for 5 minutes, and the supernatant was removed. 100 μL / well of the antibody to be tested was added, and the plate was incubated at 4°C in the dark for 1 hour. After washing three times by centrifugation at 300 g, the working concentration of PE F(ab')2-goat anti-human IgG Fc secondary antibody (Invitrogen, H10104) was added, and the plate was incubated at 4°C in the dark for 40 minutes. After washing three times by centrifugation at 300 g, the geometric mean fluorescence intensity was detected using an Invitrogen flow cytometer.

[0526] The results showed that neither antibodies Hu6 nor Hu100 bound human DLL1 and DLL4.

[0527] Test Example 4: Biacore Antibody Affinity Detection Experiment

[0528] The antibody was affinity-captured for 18 seconds using a Protein A biosensor chip (Cat.#29127556, Cytiva). Then, it was passed over the chip surface with antigens for human DLL3 (ACRO, DLL3-H52H4), monkey DLL3 (KACTUS, DLL-RM103), and mouse DLL3 (KACTUS, DLL-MM103) for 180 seconds, followed by dissociation for 600 seconds. The reaction signal was monitored in real-time using a Biacore 8K (Cytiva) instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM glycine-hydrochloric acid solution (pH 1.5) (Cat.#BR-1003-54, Cytiva). A 1:1 model was used for data fitting. The results are shown in Tables 10-1, 10-2, and 10-3.

[0529] Table 10-1. Antibody Affinity

[0530] Table 10-2. Affinity of antibody to human DLL3

[0531] Table 10-3. Affinity of antibody to human DLL3

[0532] The results showed that the humanized and chimeric antibodies of mAb100 and mAb6 specifically bound to human DLL3 with high affinity. Among them, antibody Hu6 had high affinity for DLL3 in humans, monkeys, and mice; Hu100 had high affinity for DLL3 in humans and monkeys, but did not bind to DLL3 in mice.

[0533] Test Example 5: Epitope Competition Binding Assay for Antibodies

[0534] Coat the plate with BI-764532 antibody (1 μg / mL), 100 μL / well, and incubate overnight at 4°C. Wash the plate 3 times with 250 μL / well of PBST solution. Block with 5% milk, 250 μL / well, at 37°C for 2 hours. Wash the plate 3 times with 250 μL / well of PBST solution. Add biotinylated DLL3-Strep (0.1 μg / mL, SEQ ID NO: 9). Prepare competitive antibodies, BI-764532, Hu6, and Hu100 (maximum concentration 100 μg / mL, 4-fold serial dilution), and incubate at 37°C for 1 hour. Wash the plate 6 times with 250 μL / well of PBST solution. Add streptavidin-peroxidase (1:2000 dilution) (Jackson Immuno Research, 016-030-084), 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate 6 times with 250 μL of PBST solution per well. Add 100 μL of TMB (KPL, 5120-0077) chromogenic buffer per well and incubate at room temperature for 5-10 min. Add 100 μL of 1M H2SO4 per well to stop the incubation process and read the values ​​at 450 nm using a microplate reader. The results are shown in Figure 2.

[0535] The results showed that antibodies Hu6 and Hu100 did not compete with BI-764532, indicating that antibodies Hu6 and Hu100 bind to different epitopes of BI-764532.

[0536] Test Example 6: Detection of endocytic activity of anti-DLL3 antibody

[0537] DT3C is a recombinant fusion protein composed of fragment A (toxin only) of diphtheria toxin and fragment 3C (IgG binding portion) of group G streptococci. This protein has a high affinity for the IgG portion of antibodies and enters the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, it releases the toxic DT3C, which inhibits EF2-ADP ribosylation activity, blocking protein translation and ultimately leading to cell death. DT3C that does not enter the cell does not possess cell-killing activity. The endocytic activity of the antibody is evaluated based on its cell-killing effect.

[0538] Experimental steps

[0539] a. Prepare DMS53 / DLL3 cell suspension using fresh cell culture medium RPMI1640 (GE, SH30809.01) containing 20% ​​FBS. Add 2000 cells / 50μL / well to a 96-well cell culture plate. Do not plate cells in columns 1 and 12, add only 50μL of culture medium. Incubate at 37°C for 16 hours with 5% CO2.

[0540] b. Prepare a 4× concentration DT3C (9600 nM, expressed and purified by Shanghai Panchao Biotechnology Co., Ltd.) solution using serum-free medium and filter it using a 0.22 μm filter. Prepare a 4× concentration antibody (1600 nM) using serum-free medium. Mix 80 μL of DT3C solution and 80 μL of antibody solution at a 1:1 volume ratio and incubate at room temperature for 30 minutes.

[0541] c. Dilute the mixture five-fold with serum-free medium to obtain nine concentrations, with the tenth point being pure medium.

[0542] d. Add 50 μL of diluted antibody to the cells and incubate in an incubator for three days.

[0543] e. Add 50 μL of CTG to each well. Luminescent Cell Viability Assay (Promega, G7573), incubated at room temperature in the dark for 10 minutes, and chemiluminescence was read on a Victor3.

[0544] The results are shown in Table 11 and Figure 3 below.

[0545] Table 11. Cell killing caused by antibody endocytosis

[0546] The results showed that both antibodies Hu6 and Hu100 could be endocytosed by cells.

[0547] Test Example 7: FACS Detection of ADC Binding at the Cellular Level

[0548] hDLL3 / CHO-s and cynoDLL3 / CHO-s cells expressing DLL3 were prepared into 1×10⁻⁶ cells using FACS buffer (1% BSA + pH 7.4 PBS). 6 100 μL / well of cell suspension was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add 100 μL / well of the target ADC at different concentrations. Incubate at 4°C in the dark for 1 hour. After washing three times by centrifugation at 300g, add the working concentration of APC anti-human IgG Fc (BioLegend, 410712) or PE F(ab')2-goat anti-human IgG (Invitrogen, H10104) and incubate at 4°C in the dark for 40 minutes. After washing three times by centrifugation at 300g, detect the geometric mean fluorescence intensity on an Invitrogen flow cytometer and calculate the EC50 of the antibody binding to DLL3-expressing cells. 50 Values. The results are shown in Table 12, Figure 4A and Figure 4B.

[0549] Table 12. Binding activity of antibodies to DLL3 expressed by cells

[0550] The results showed that the ADCs disclosed in this study could specifically bind to human and monkey DLL3 expressed in cells, and the binding of ADCs was basically unaffected by the increase of DAR value.

[0551] Test Example 8: Biacore's Experiment on Detecting ADC Affinity

[0552] The antibody was affinity-captured for 18 seconds using a Protein A biosensor chip (Cat.#29127556, Cytiva), followed by flow of antigen-human DLL3 (ACRO, DLL3-H52H4) and monkey DLL3 (KACTUS, DLL-RM103) onto the chip surface for 180 seconds, and then dissociated for 600 seconds. Binding and dissociation curves were obtained by real-time monitoring of the reaction signal using a Biacore 8K (Cytiva) instrument. After dissociation in each experimental cycle, the biosensor chip was regenerated by washing with 10 mM glycine-hydrochloric acid solution (pH 1.5) (Cat.#BR-1003-54, Cytiva). A 1:1 model was used for data fitting. The results are shown in Table 13.

[0553] Table 13. Affinity of ADC to DLL3 of different species

[0554] The results showed that all the ADCs disclosed in this study could specifically bind to the human and monkey DLL3 protein. Furthermore, the binding of the ADCs was essentially unaffected by increasing DAR values.

[0555] Test Example 9: Cytotoxicity of ADCs against Different DLL3 Expression Levels

[0556] The experimental steps are as follows:

[0557] a. Prepare a cell suspension using fresh cell culture medium containing 10% FBS, and add 135 μL per well to a 96-well cell culture plate (Corning, 3903). Do not seed cells in columns 1 and 12; add only 135 μL of culture medium to each column. Incubate at 37°C with 5% CO2 for 16 hours.

[0558] b. Prepare the ADC sample into a working solution (10× concentration) using PBS. Use this as the initial concentration and then perform serial dilutions with PBS at the corresponding multiples. Add 15 μL of 10× concentration ADC solution to each well and incubate at 37°C for 6 days with 5% CO2.

[0559] c. Add 70 μL of CTG (Promega, G7573) to each well and incubate at room temperature in the dark for 10 minutes. Read the chemiluminescence on a Victor3 and process the data using a GraphPad Prism5. Plot the data with antibody or ADC concentration as the X-axis and light intensity as the Y-axis.

[0560] The different cell bed densities, working solution concentrations (10× concentration) in the first well, and dilution factors are shown in Table 14.

[0561] Table 14. Cell plating density and working solution concentration in the first well

[0562] The cells used in the experiment are as follows:

[0563] H1184 (+++) was purchased from ATCC, CRL-5858;

[0564] The DMS53(++) was purchased from ATCC, CRL-2062;

[0565] SK-MEL3(+) was purchased from ATCC, HTB-69;

[0566] CHO-K1(-) was purchased from ATCC, CCL-61.

[0567] The "+" indicates the expression level of DLL3, and the "-" indicates that DLL3 is not expressed.

[0568] The results are shown in Table 15 and Figures 5A to 5D.

[0569] Table 15. Killing activity of ADCs against cells with different DLL3 expression levels

[0570] The results showed that ADC-1, ADC-2 and ADC-3 had strong target cell killing activity and could kill SK-MEL3, DMS53 and H1184 cells expressing DLL3, but had no killing effect on DLL3-negative CHO-K1 cells.

[0571] Test Example 10: Bystander Killing Activity Experiment

[0572] DMS53 / DLL3high (DMS53 cells stably transfected with DLL3) and U-2OS (ATCC, HTB-96) cells were cultured in RPMI 1640 + 20% FBS + 1×Glutamax and McCoy's 5A + 10% FBS, respectively. Cells were trypsinized, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, and the cells were resuspended in RPMI 1640 + 20% FBS + 1×Glutamax. After cell counting, the DMS53 / DLL3high cell density was adjusted to 9 × 10⁶ cells / year.4 U-2OS cell density was adjusted to 3 × 10⁶ cells / mL. 4 Cells / mL. Add 500 μL of DMS53 / DLL3high cells and 500 μL of U-2OS cells to each well of a 12-well plate. Add 500 μL of U-2OS cells and 500 μL of RPMI 1640 + 20% FBS + 1× Glutamax medium to each well of a 12-well plate. Incubate at 37°C with 5% CO2 for 24 hours. Prepare a 40× intermediate solution (200 nM). Add 25 μL of ADC sample to each well of the 12-well plate. Set up a solvent control group. Incubate at 37°C with 5% CO2 for 6 days. Digest cells in the 12-well plate with trypsin, neutralize with fresh medium, add 20 μL of cells to 20 μL of trypan blue, and count. Centrifuge cells at 1000 rpm for 3 minutes and discard the supernatant. Wash cells once with 100 μL of FACS buffer, centrifuge at 1500 rpm for 3 minutes, and discard the supernatant. Resuspend the cells in 100 μL of FACS buffer, add 2 μg / mL of anti-DLL3 positive antibody, and incubate on ice for 60 minutes. Wash once with FACS buffer, centrifuge at 1500 rpm for 3 minutes, add secondary antibody APC anti-human IgG Fc (100x), incubate for 30 minutes, and wash once with FACS buffer. Resuspend the cells in 200 μL of FACS buffer and detect them using FACS. FlowJo was used to analyze the flow cytometry data to obtain the DMS53 / DLL3high ratio and calculate the total number of DMS53 / DLL3high and U-2OS. GraphPad Prism5 was used to plot the data, with different samples on the X-axis and the calculated cell count on the Y-axis. The results are shown in Figure 6.

[0573] The results showed that ADC-1, ADC-2 and ADC-3 had significant bystander cytotoxic effects.

[0574] In vivo bioactivity evaluation

[0575] Test Example 11-1: In vivo efficacy evaluation of DMS53 cell CDX mouse model

[0576] Human small cell lung cancer DMS53 cells (5×10) 6 Each animal was injected subcutaneously with 200 μL of ATCC (CRL-2062) containing 50% Matrigel. The injection was administered subcutaneously to the right rib area of ​​Balb / c. Seven days after injection, when the tumor volume reached approximately 220 mm², the tumor was monitored. 3After removing mice by weight, excessively large or small tumors, and other factors, mice were randomly assigned to groups of eight based on tumor volume. Drug administration began on the same day. ADC was administered intraperitoneally at a dose of 1.5 mg / kg, once weekly for a total of two administrations. Drug administration was discontinued after the second administration, and tumor growth was continuously monitored. Tumor volume and body weight were measured twice weekly, and data were recorded. Data were recorded using Excel statistical software: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT (number of animals per group); graphs were plotted using GraphPad Prism software, and statistical analysis was performed using either two-way ANOVA or one-way ANOVA.

[0577] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0578] The 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.

[0579] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[0580] The results are shown in Table 16 and Figure 7.

[0581] Table 16. Efficacy of ADC on DMS53 xenografts in tumor-bearing nude mice

[0582] The results showed that at a dose of 1.5 mpk, both ADC-1 and ADC-4 significantly inhibited the growth of subcutaneous DMS53 cell-derived tumors. At doses of 0.5 mpk and 1.5 mpk, both ADC-2 and ADC-5 significantly inhibited the growth of subcutaneous DMS53 cell-derived tumors.

[0583] Test Example 11-2: In vivo efficacy evaluation of DMS53 cell CDX mouse model

[0584] Human small cell lung cancer DMS53 cells (5×10) 6 Each animal was injected subcutaneously with 200 μL of ATCC (CRL-2062) containing 30% Matrigel. The injection was administered subcutaneously to the right rib area of ​​Balb / c. Fifteen days after injection, when the tumor volume reached approximately 180 mm², the tumor was monitored. 3After removing mice by weight, excessively large or small tumors, and other factors, mice were randomly assigned to groups of eight based on tumor volume. Drug administration began on the same day. ADC was administered intraperitoneally at doses of 1.5 mg / kg or 0.5 mg / kg, once weekly for a total of two administrations. Drug administration was discontinued after the second administration, and tumor growth was continuously monitored. Tumor volume and body weight were measured twice weekly, and data were recorded. Data were recorded using Excel statistical software: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT (number of animals per group); graphs were created using GraphPad Prism software, and statistical analysis was performed using either two-way ANOVA or one-way ANOVA.

[0585] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0586] The 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.

[0587] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[0588] The results are shown in Table 17 and Figure 8.

[0589] Table 17. Efficacy of ADC on DMS53 xenografts in tumor-bearing nude mice

[0590] The results showed that ADC-3 at doses of 0.5 mpk and 1.5 mpk, and ADC-1 at a dose of 1.5 mpk, could significantly inhibit the growth of subcutaneously transplanted DMS53 cells.

[0591] Test Example 12: In vivo efficacy evaluation of NCI-H1184 cell CDX mouse model

[0592] NCI-H1184 cells (6×10) 6 One dose of 50% MatrixGel (200 μL / mouse) containing ATCC (CRL-5858) was injected subcutaneously into the right rib area of ​​80 NDG mice. Thirteen days after inoculation, when the average tumor volume was ~185 mm, 3After removing mice by weight, excessively large or small tumors, and other factors, mice were randomly divided into 7 groups of 9 mice each, based on tumor volume. Drug administration began on the same day. Antibodies were administered intraperitoneally for 21 days. Tumor volume and body weight were measured twice weekly, and data were recorded. Data were recorded using Excel statistical software: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT (number of animals per group); GraphPad Prism software was used for graphing, and two-way ANOVA or one-way ANOVA was used for statistical analysis.

[0593] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0594] The 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.

[0595] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[0596] The results are shown in Table 18 and Figure 9.

[0597] Table 18. Efficacy of ADC on H1184 xenografts in tumor-bearing nude mice

[0598] The results showed that both ADC-2 and ADC-3 could significantly inhibit the growth of NCI-H1184 tumors.

[0599] Test Example 13: Rat PK Study

[0600] The single-dose pharmacokinetics of ADC molecules were studied in SD rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0601] Rat ADC molecules were administered intravenously at 3 mg / kg (n = 4 / group). Blood samples were collected from the fundus veins of rats at 5 min, 8 h, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 21 d, and 28 d after administration. Collected blood samples were left at room temperature for half an hour until agglutination, then centrifuged at 1000g for 15 minutes at 4°C, and serum was collected. Bioanalytical measurements were performed on rat serum samples using the HTRF method. Biotin-labeled DLL3 capture antibody and biotin-labeled antitoxin antibody capture toxin were used. Quantitative analysis of the analyte content was performed using a four-parameter model curve of standards. Pharmacokinetic parameters were analyzed using a standard non-compartmental model in WinNonlin software (6.4). Experimental results are shown in Table 19 and Figures 10A to 10C.

[0602] Table 19. Rat Pharmacokinetics of ADC Molecules

[0603] The results showed that the total antibody measured in vivo by the ADC molecules had a similar AUC to that of the complete ADC, indicating that the complete ADC structures of the three ADC molecules were all stable.

[0604] Test Example 14: Monkey vs. Monkey Study

[0605] The single-dose pharmacokinetics of ADC molecules were studied in cynomolgus monkeys (Suzhou Xishan Zhongke Experimental Animal Co., Ltd.).

[0606] ADC molecules were administered intravenously at 10 mg / kg (n = 3 / group) at 5 min, 8 h, 1 d, 1.5 d, 2 d, 3 d, 4 d, 7 d, 10 d, 14 d, 21 d, and 28 d. Collected blood samples were left at room temperature for half an hour until agglutination, then centrifuged (4 ℃, 2600 g, 10 min), and the supernatant serum was collected and immediately stored at -60 ℃. Serum bioanalytical measurements were performed using the DELFIA method, and the content of the analyte was quantitatively analyzed using a four-parameter model curve of the standards. Pharmacokinetic parameters were analyzed using a standard non-compartmental model in WinNonlin software (6.4). Experimental results are shown in Table 20 and Figure 11.

[0607] Table 20. Pharmacokinetics of ADC molecules in monkeys

[0608] The results showed that after intravenous administration of 10 mpk of ADC-2 to cynomolgus monkeys, the half-lives of total antibody and intact ADC were 9.9 ± 1.1 days and 8.5 ± 0.1 days, respectively; the AUCs of total antibody and intact ADC were 27405 μg / mL*h and 25702 μg / mL*h, respectively. The half-lives of total antibody and intact ADC in cynomolgus monkeys were essentially the same, and the AUC ratio of intact ADC to total antibody was 94%, indicating that ADC-2 had good stability in cynomolgus monkeys.

[0609] III. Preparation Examples - Anti-DLL3 Antibody-Drug Conjugate Formulation

[0610] SEC size exclusion chromatography:

[0611] An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0612] SEC% polymer (SEC polymer content percentage) = A polymer / A total × 100% (A polymer is the peak area of ​​the polymer peak in the sample, and A total is the sum of the peak areas of all peaks). ΔSEC% polymer = SEC% polymer of the formulation after stability test - SEC% polymer of the formulation before stability test.

[0613] Instrument used for SEC determination: Agilent HPLC 1260.

[0614] Pillars: Waters, BioResolve TM SEC mAb 2.5μm 7.8×300mm Column.

[0615] R-CE capillary gel electrophoresis:

[0616] An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and then separated according to the molecular weight of the sample under a certain voltage.

[0617] R-CE% (R-CE content percentage) = Amain peak / Atotal × 100% (Amain peak is the peak area of ​​the light chain main peak + heavy chain main peak in the sample, and Atotal is the sum of the peak areas of all peaks). ΔR-CE% = R-CE% of the formulation after stability test - R-CE% of the formulation before stability test.

[0618] The instrument used for CE testing was a Beckman capillary electrophoresis apparatus, model PA800 plus.

[0619] Free toxins:

[0620] Based on the principle of reversed-phase separation, protein precipitation was followed by UPLC-UV analysis to detect the free toxin content in the supernatant. The concentration of the corresponding free toxin was calculated by substituting the sample peak area into the linear regression equation of the standard.

[0621] Instrument used for free toxin determination: Waters Acquity H Class.

[0622] Osmotic pressure measurement:

[0623] The freezing point method for determining osmotic pressure is based on the principle that the freezing point depression is directly proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and converts the electrical charge into osmotic pressure.

[0624] Instrument used for osmotic pressure measurement: Loser, model OM815.

[0625] protein

[0626] The concentration of the anti-DLL3 antibody drug conjugate (prepared with reference to the method of ADC-2 above, DAR: approximately 8, hereinafter referred to as "protein") disclosed herein is calculated based on the protein concentration, that is, based on the concentration of the anti-DLL3 antibody portion in the anti-DLL3 antibody drug conjugate.

[0627] Because the toxin in the anti-DLL3 antibody-drug conjugate absorbs at the characteristic absorption wavelength of proteins at 280 nm, and also absorbs at 370 nm, the protein concentration is calculated using the following formula: A 280nm =(C drug ×E drug-280 +C mAb ×E mAb-280 )×l A 370nm =C drug ×E drug-370 ×l

[0628] Pick

[0629] Right now:

[0630] In the formula, A 280nm The average absorbance of a single sample of the test solution at a wavelength of 280 nm when the optical path length is 1 cm;

[0631] A 370nm The average absorbance of a single sample of the test solution at a wavelength of 370 nm when the optical path length is 1 cm;

[0632] E mAb-280 The mass extinction coefficient of the protein at a wavelength of 280 nm is 1.52 g⁻¹ cm⁻¹ L.

[0633] E drug-280 The mass extinction coefficient of the toxin at a wavelength of 280 nm is 5.17 g⁻¹ cm⁻¹ L.

[0634] E drug-370 The mass extinction coefficient of the toxin at a wavelength of 370 nm is 17.89 g⁻¹cm⁻¹L.

[0635] R: The ratio of the toxin extinction coefficient at 370nm to 280nm is 3.46;

[0636] C mAb Protein concentration, mg / mL;

[0637] l: Optical path length, cm (the optical path length here is 1 cm).

[0638] If the test solution is diluted, the protein concentration is: C (mg / mL) = C mAb ×N, where N is the dilution factor.

[0639] Protein concentration measurement instrument: UV-Vis spectrophotometer, model: Nano Drop 2000.

[0640] Formulation Example 1. Screening of pH and Buffer Systems

[0641] A formulation containing 20 mg / mL protein, 80 mg / mL sucrose, and 0.4 mg / mL polysorbate 80 (PS80) was prepared using the buffer system shown in Table 21. Forced degradation studies (incubation at 40°C for 1 month) were conducted on the samples, and the effects of different pH values ​​and buffer systems on protein stability were investigated using SEC and R-CE as evaluation indicators.

[0642] The results are shown in Table 21. After one month of storage at 40℃, the His-HCl pH 5.5 group showed the least increase in SEC polymers, while the His-HCl pH 6.0 group showed the least decrease in R-CE purity. Therefore, the His-HCl buffer system is superior to the SA buffer system, and 10mM His-HCl at pH 5.5-6.0 also exhibits good stability.

[0643] Table 21. Screening Results of pH and Buffer Systems

[0644] Note: His-HCl represents histidine-histidine hydrochloride; SA represents succinic acid-sodium succinate; 40℃ M1 represents storage at 40℃ for 1 month, the same applies below.

[0645] Formulation Example 2. pH Range Confirmation

[0646] Formulas containing 20 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80 (PS80), and 0.05 mg / mL disodium ethylenediaminetetraacetate dihydrate (Na2EDTA·2H2O) were prepared using 10 mM His-HCl buffers with different pH values ​​as shown in Table 22. Accelerated stability studies (6 months at 25°C) were conducted on the samples, using SEC and R-CE as evaluation indicators to investigate the effect of different buffer pH values ​​on protein stability.

[0647] The results are shown in Table 22. SEC and R-CE results showed that the differences between the groups using 10mM His-HCl at pH 5.0-6.0 were small, and all groups exhibited good acceleration stability.

[0648] Table 22. Results of pH Range Confirmation Experiment

[0649] Note: 25℃ M6 means 6 months at 25℃, the same applies below.

[0650] Formulation Example 3. Buffer Ionic Strength Screening 1

[0651] Formulas containing 20 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL PS80, and 0.05 mg / mL Na2EDTA·2H2O were prepared using His-HCl pH 5.5 buffers with different ionic strengths as shown in Table 23. Forced degradation studies were conducted on the samples (40℃ for 4 weeks), and the effect of buffer systems with different ionic strengths on protein stability was investigated using R-CE as the evaluation index.

[0652] The results are shown in Table 23. After being placed at 40℃ for 4 weeks, the R-CE results showed that the formulations prepared with 5mM to 20mM His-HCl pH5.5 buffers had small differences between groups and exhibited good high-temperature stability.

[0653] Table 23. Results of the buffer solution ionic strength screening experiment 1

[0654] Note: 40℃ W4 means placed at 40℃ for 4 weeks, the same applies below.

[0655] Formulation Example 4. Buffer Ionic Strength Screening 2

[0656] Formulas containing 20 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL PS80, and 0.05 mg / mL Na2EDTA·2H2O were prepared using His-HCl pH 5.5 buffers with different ionic strengths as shown in Table 24. Long-term stability studies were conducted on the samples (stored at 2–8°C for 6 months), using SEC and R-CE as evaluation indicators to investigate the effect of buffer systems with different ionic strengths on protein stability.

[0657] The results are shown in Table 24. After being stored at 2–8°C for 6 months, SEC and R-CE results showed that the preparations prepared with 10 mM to 30 mM His-HCl pH 5.5 buffer showed little difference between groups.

[0658] Table 24. Results of the buffer solution ionic strength screening experiment 2

[0659] Note: 2~8℃ M6 means 6 months at 2~8℃, the same applies below.

[0660] Formulation Example 5. Effect of excipient Na2EDTA·2H2O on formulation stability

[0661] Formulations containing 20 mg / mL protein, 80 mg / mL sucrose, and 0.4 mg / mL PS80, as shown in Table 25, were prepared using 10 mM His-HCl pH 5.5 buffer. The formulations without Na2EDTA·2H2O and with 0.05 mg / mL Na2EDTA·2H2O were also prepared. Forced degradation studies (incubation at 40°C for 1 month) were conducted on the samples, and the effect of Na2EDTA·2H2O on the stability of the formulations was investigated using SEC as the evaluation index.

[0662] The results are shown in Table 25. After being stored at 40℃ for 1 month, the SEC results showed no significant difference between the formulation groups without Na2EDTA·2H2O and those with 0.05 mg / mL Na2EDTA·2H2O.

[0663] Table 25. Experimental results on the effect of Na2EDTA·2H2O on formulation stability

[0664] Note: N / A means not added.

[0665] Formulation Example 6. Screening of excipient Na2EDTA·2H2O concentration

[0666] Formulations containing 20 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL PS80, and different concentrations of Na2EDTA·2H2O (as shown in Table 26) were prepared using 10 mM His-HCl pH 5.5 buffer. Forced degradation studies (40°C for 4 weeks) were conducted on the samples, and the effects of different Na2EDTA·2H2O concentrations on the stability of the formulations were investigated using SEC and R-CE as evaluation indicators.

[0667] The results are shown in Table 26. After being placed at 40℃ for 4 weeks, SEC and R-CE results showed no significant differences among the formulation groups with Na2EDTA·2H2O concentrations ranging from 0.01 mg / mL to 0.1 mg / mL.

[0668] Table 26. Screening Results of Na2EDTA·2H2O Concentration

[0669] Formulation Example 7. Surfactant Concentration Screening

[0670] Formulations containing 20 mg / mL protein, 80 mg / mL sucrose, 0.05 mg / mL Na₂EDTA·2H₂O, and different concentrations of PS80 (as shown in Table 27) were prepared using 10 mM His-HCl pH 5.5 buffer. Forced degradation studies (40°C for 4 weeks) were conducted on the samples, and the effects of different surfactant concentrations on protein stability were investigated using SEC and R-CE as evaluation indicators.

[0671] The results are shown in Table 27. After 4 weeks of storage at 40°C, SEC and R-CE results showed no significant differences among the formulation groups with PS80 concentrations ranging from 0.02 mg / mL to 0.6 mg / mL.

[0672] Table 27. Results of Surfactant Concentration Screening

[0673] Formulation Example 8. Screening of Sucrose Concentration

[0674] Formulations containing 20 mg / mL protein, 0.4 mg / mL PS80, 0.05 mg / mL Na2EDTA·2H2O, and different concentrations of sucrose (as shown in Table 28) were prepared using 10 mM His-HCl pH 5.5 buffer. Forced degradation studies (40°C for 4 weeks) were conducted on the samples, and the effects of different sucrose concentrations on the stability of the formulations were investigated using SEC and R-CE as evaluation indicators.

[0675] The results are shown in Table 28. After 4 weeks of storage at 40℃, SEC and R-CE results showed no significant differences among the formulation groups with sucrose concentrations ranging from 30 mg / mL to 100 mg / mL. When the sucrose concentration was 80 mg / mL, the osmotic pressure of the formulation was 294 mOsm, which met the isotonic requirement of the formulation. Therefore, the preferred sucrose concentration was 80 mg / mL.

[0676] Table 28. Results of Sucrose Concentration Screening

[0677] Formulation Example 9. Protein Concentration Screening

[0678] Formulations with different protein concentrations, 80 mg / mL sucrose, 0.4 mg / mL PS80, and 0.05 mg / mL Na2EDTA·2H2O (as shown in Table 29) were prepared using 10 mM His-HCl pH 5.5 buffer. Forced degradation studies (40°C for 4 weeks) were conducted on the samples, and the effect of different protein concentrations on protein stability was investigated using R-CE as the evaluation index.

[0679] The results are shown in Table 29. After 4 weeks of storage at 40℃, the R-CE results showed that there were little difference between the groups for protein concentrations ranging from 5 mg / mL to 30 mg / mL.

[0680] Table 29. Protein Concentration Screening Results

[0681] Formulation Example 10. Formulation Confirmation 1 (Solution)

[0682] A formulation containing 20 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL PS80, and 0.05 mg / mL Na2EDTA·2H2O was prepared using 10 mM His-HCl pH 5.5 buffer. Long-term stability studies were conducted on the samples (stored at 2–8 °C for 3 months), using SEC, R-CE, and free toxins as evaluation indicators to assess the stability of the formulation.

[0683] The results are shown in Table 30. After being stored at 2–8°C for 3 months, there were no significant changes in SEC, R-CE, and free toxins, indicating that the protein stability of this formulation is good.

[0684] Table 30. Long-term stability results of the prescription

[0685] Note: 2~8℃ M3 means 3 months at 2~8℃, the same applies below.

[0686] Formulation Example 11. Formulation Confirmation 2 (Solution)

[0687] A formulation containing 20 mg / mL protein, 80 mg / mL sucrose, 0.4 mg / mL PS80, and 0.05 mg / mL Na2EDTA·2H2O was prepared using 10 mM His-HCl pH 5.5 buffer. Long-term stability studies were conducted on the samples (stored at 2–8 °C for 12 months), using SEC, R-CE, and free toxins as evaluation indicators to assess the stability of the formulation.

[0688] The results are shown in Table 31. After being stored at 2–8°C for 12 months, the changes in SEC, R-CE, and free toxins were all within acceptable ranges, indicating that the protein stability of this formulation was good.

[0689] Table 31. Long-term stability results of the prescription

[0690] Note: 2~8℃ M12 means 2~8℃ for 12 months.

[0691] Formulation Example 12. Formulation Confirmation 3 (Lyophilized Formulation)

[0692] Stock solutions of 20 mg / mL protein, 40 mg / mL sucrose, 9 mg / mL glycine, and 0.2 mg / mL PS80 were prepared using 30 mM His-HCl pH 5.5 buffer and then lyophilized. Forced degradation stability studies (40°C for 4 weeks) and long-term stability studies (2–8°C for 3 months) were conducted on the samples. Appearance, SEC, and R-CE were used as evaluation indicators to assess the stability of the formulation.

[0693] The freeze-drying process is shown in Table 32. After freeze-drying, the surface of the powder cake is flat and does not collapse.

[0694] Table 32. Freeze-drying procedures for lyophilized formulations

[0695] The results are shown in Table 33. After being placed at 40℃ for 4 weeks and at 2-8℃ for 3 months, there were no significant changes in appearance, SEC, and R-CE after reconstitution, indicating that the protein in this formulation has good stability.

[0696] Table 33. Stability results of lyophilized formulations

Claims

1. A pharmaceutical composition comprising an anti-DLL3 antibody-drug conjugate and a buffer, wherein: The anti-DLL3 antibody-drug conjugate has the following structure: in: n is between 1 and 10; Pc is an anti-DLL3 antibody, which contains a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable regions HCDR1, HCDR2 and HCDR3 contain the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, and the light chain variable regions LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively. The buffer is a histidine buffer or a succinate buffer; Preferably, the buffer is a histidine-histidine hydrochloride buffer or a succinic acid-sodium succinate buffer. More preferably, the buffer is a histidine-histidine hydrochloride buffer.

2. The pharmaceutical composition according to claim 1, wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:55; Preferably, the anti-DLL3 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO:

61.

3. The pharmaceutical composition according to claim 1 or 2, wherein n is 3 to 8; preferably, n is 6 to 8; more preferably, n is about 8.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of the anti-DLL3 antibody drug conjugate is from 1 mg / mL to 50 mg / mL; Preferably, the concentration of the anti-DLL3 antibody-drug conjugate is from 5 mg / mL to 30 mg / mL; More preferably, the concentration of the anti-DLL3 antibody-drug conjugate is from 15 mg / mL to 25 mg / mL; Most preferably, the concentration of the anti-DLL3 antibody-drug conjugate is about 20 mg / mL.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration of the buffer is from 1 mM to 50 mM; Preferably, the concentration of the buffer is from 5 mM to 40 mM; More preferably, the concentration of the buffer is 5 mM to 30 mM, or 5 mM to 35 mM; More preferably, the concentration of the buffer is 8 mM to 12 mM, or 25 mM to 35 mM; Most preferably, the concentration of the buffer is about 9.2 mM, about 10 mM, about 27.8 mM, or about 30 mM.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pH of the pharmaceutical composition is 4.5 to 8.0; Preferably, the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pH of the pharmaceutical composition is 5.2 to 5.8; Most preferably, the pH of the pharmaceutical composition is about 5.6 or about 5.

5.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition comprises a surfactant; Preferably, the surfactant is polysorbate; More preferably, the surfactant is polysorbate 80.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the concentration of the surfactant is from 0.01 mg / mL to 2 mg / mL; Preferably, the concentration of the surfactant is from 0.02 mg / mL to 0.6 mg / mL; More preferably, the concentration of the surfactant is from 0.1 mg / mL to 0.6 mg / mL; More preferably, the concentration of the surfactant is from 0.3 mg / mL to 0.5 mg / mL, or from 0.1 mg / mL to 0.3 mg / mL; Most preferably, the concentration of the surfactant is about 0.4 mg / mL or about 0.2 mg / mL.

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

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the concentration of the sugar is from 10 mg / mL to 120 mg / mL; Preferably, the concentration of the sugar is from 30 mg / mL to 100 mg / mL; More preferably, the concentration of the sugar is from 65 mg / mL to 95 mg / mL, or from 32 mg / mL to 48 mg / mL; Most preferably, the concentration of the sugar is about 80 mg / mL or about 40 mg / mL.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pharmaceutical composition further comprises excipients; Preferably, the excipients are ethylenediaminetetraacetic acid or its salt, ethylenediaminetetraacetic acid hydrate or its salt, glycine, DTPA, arginine hydrochloride, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride or calcium chloride; More preferably, the excipient is a sodium salt of ethylenediaminetetraacetic acid hydrate or glycine; Most preferably, the excipient is disodium ethylenediaminetetraacetate dihydrate or glycine.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the excipient is disodium ethylenediaminetetraacetate dihydrate, and the concentration of the excipient is from 0.01 mg / mL to 1 mg / mL; Preferably, the concentration of the excipient is from 0.01 mg / mL to 0.5 mg / mL; More preferably, the concentration of the excipient is from 0.01 mg / mL to 0.1 mg / mL; Most preferably, the concentration of the excipient is about 0.05 mg / mL.

13. The pharmaceutical composition according to any one of claims 1 to 11, wherein the excipient is glycine, and the concentration of the excipient is from 5 mg / mL to 15 mg / mL; Preferably, the concentration of the excipient is from 7 mg / mL to 11 mg / mL; More preferably, the concentration of the excipient is about 9 mg / mL.

14. The pharmaceutical composition according to any one of claims 1 to 12, comprising the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 1 mg / mL to 50 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and (e) a buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0; Preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL, (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.5 mg / mL, and (e) a 5 mM to 40 mM histidine-histidine hydrochloride buffer or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.6 mg / mL, (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and (e) a 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL, (b) Polysorbate 80 at concentrations of 0.3 mg / mL to 0.5 mg / mL, (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and (e) an 8 mM to 12 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8; Most preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.4 mg / mL of polysorbate 80, (c) Approximately 80 mg / mL of sucrose, (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) about 9.2 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.6; or (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.4 mg / mL of polysorbate 80, (c) Approximately 80 mg / mL of sucrose, (d) Approximately 0.05 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.

5.

15. The pharmaceutical composition according to any one of claims 1 to 10, comprising the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 1 mg / mL to 50 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, and (d) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0; Preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL, (c) Sucrose at concentrations from 30 mg / mL to 100 mg / mL, and (d) A 5 mM to 40 mM histidine-histidine hydrochloride buffer or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.6 mg / mL, (c) Sucrose at concentrations from 30 mg / mL to 100 mg / mL, and (d) A 5 mM to 30 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL, (b) Polysorbate 80 at concentrations of 0.3 mg / mL to 0.5 mg / mL, (c) Sucrose at concentrations of 65 mg / mL to 95 mg / mL, and (d) An 8 mM to 12 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8; Most preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.4 mg / mL of polysorbate 80, (c) Approximately 80 mg / mL of sucrose, and (d) Approximately 9.2 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is approximately 5.6; or (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.4 mg / mL of polysorbate 80, (c) Approximately 80 mg / mL of sucrose, and (d) About 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.

5.

16. The pharmaceutical composition according to any one of claims 1 to 11, 13, comprising the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 1 mg / mL to 50 mg / mL, (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and (e) a buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0; Preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.02 mg / mL to 0.6 mg / mL, (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL, (d) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and (e) a 5 mM to 40 mM histidine-histidine hydrochloride buffer or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations ranging from 5 mg / mL to 30 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.6 mg / mL, (c) Sucrose at concentrations of 30 mg / mL to 100 mg / mL, (d) Glycine at concentrations of 5 mg / mL to 15 mg / mL, and (e) a 5 mM to 40 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at concentrations of 15 mg / mL to 25 mg / mL, (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.3 mg / mL, (c) Sucrose at concentrations of 32 mg / mL to 48 mg / mL, (d) Glycine at concentrations of 7 mg / mL to 11 mg / mL, and (e) a 25 mM to 35 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is 5.2 to 5.8; Most preferably, the pharmaceutical composition comprises the following components: (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.2 mg / mL of polysorbate 80, (c) Approximately 40 mg / mL of sucrose, (d) Approximately 9 mg / mL of glycine, and (e) a histidine-histidine hydrochloride buffer of about 27.8 mM, wherein the pH of the pharmaceutical composition is about 5.5; or (a) The anti-DLL3 antibody-drug conjugate at a concentration of approximately 20 mg / mL, (b) Approximately 0.2 mg / mL of polysorbate 80, (c) Approximately 40 mg / mL of sucrose, (d) Approximately 9 mg / mL of glycine, and (e) about 30 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.

5.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein it is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation, or an intramuscular injection preparation; preferably an intravenous injection preparation.

18. A lyophilized formulation, wherein the lyophilized formulation, upon reconstitution, can form a pharmaceutical composition according to any one of claims 1 to 16.

19. A method of treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 16, or the lyophilized formulation of claim 18; Preferably, the disease is a tumor or cancer; More preferably, the diseases are selected from: lung cancer, melanoma, medullary thyroid carcinoma, glioblastoma, neuroendocrine tumors, prostate cancer, glioma, thyroid cancer, head and neck cancer, brain cancer, glioblastoma multiforme, neuroblastoma, central nervous system cancer, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, malignant pleural mesothelioma, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, testicular cancer, adrenal cancer, and skin cancer; Most preferably, the disease is small cell lung cancer.

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