WO2025131024A1 - 一种包含特异性结合dll3和cd3的双特异性抗体的药物组合物 - Google Patents

一种包含特异性结合dll3和cd3的双特异性抗体的药物组合物 Download PDF

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WO2025131024A1
WO2025131024A1 PCT/CN2024/140832 CN2024140832W WO2025131024A1 WO 2025131024 A1 WO2025131024 A1 WO 2025131024A1 CN 2024140832 W CN2024140832 W CN 2024140832W WO 2025131024 A1 WO2025131024 A1 WO 2025131024A1
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Prior art keywords
pharmaceutical composition
dll3
concentration
histidine
bispecific antibody
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French (fr)
Inventor
刘玥
杨健健
田晨敏
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Shanghai Hengrui Pharmaceutical Co Ltd
Jiangsu Hengrui Pharmaceutical Co Ltd
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Shanghai Hengrui Pharmaceutical Co Ltd
Jiangsu Hengrui Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure belongs to the field of biotechnology pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to DLL3 and CD3.
  • Small cell lung cancer is a relatively malignant type of lung cancer, accounting for 10%-15% of all lung cancer cases. Small cell lung cancer tumors grow rapidly, are easy to metastasize, and have a 5-year survival rate of less than 7%.
  • the treatment of small cell lung cancer is relatively limited, mainly chemical drugs, such as chemotherapy combined with platinum/etoposide. Patients with small cell lung cancer respond well to chemotherapy in the early stages, but are very likely to develop drug resistance and relapse.
  • Immunotherapy in recent years, such as PD-L1 antibodies and PD1 antibodies has a certain effect on SCLC patients, but the effective rate is about 15%. At present, specific targeted therapy drugs have not yet been developed.
  • DLL3 is a ligand that inhibits Notch. Under normal conditions, DLL3 is located on the Golgi apparatus. In cancer cells (such as small cell lung cancer cells), DLL3 is expressed on the cell surface. DLL3 binds to Notch in a cis manner, hindering cell-cell binding and Notch endocytosis in target cells, thereby inhibiting the Notch signaling pathway and promoting the growth of tumor cells. DLL3 is mainly expressed in neural or neuroendocrine tumors, including small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, small cell bladder cancer, multiforme glioma, metastatic castration-resistant prostate cancer, melanoma, etc., especially SCLC. More than 80% of SCLC have positive expression of DLL3, while normal lung cancer tissue and adjacent tissues do not express it. This difference in expression makes DLL3 a very potential therapeutic target for the treatment of SCLC.
  • CD3 is a homotypic or heterotypic dimeric antigen expressed on T cells. Functional CD3 is formed by dimerization of two of four different chains: ⁇ , ⁇ , ⁇ , and ⁇ . CD3 dimer arrangements include ⁇ / ⁇ , ⁇ / ⁇ , and ⁇ / ⁇ . CD3 binds to the T cell receptor complex (TCR) and is required for T cell activation. Therefore, anti-CD3 antibodies that activate T cells have been proposed for the treatment of cancer. However, the administration of anti-CD3 antibodies may trigger T cell activation and related cytokine release, and excessive cytokine release leads to severe cytokine release syndrome (CRS), which is an important challenge for the clinical use of anti-CD3 antibodies.
  • TCR T cell receptor complex
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a bispecific antibody that specifically binds DLL3 and CD3 and a buffer.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a bispecific antibody that specifically binds to DLL3 and CD3 and a buffer, wherein:
  • the bispecific antibody that specifically binds to DLL3 and CD3 comprises a first chain having a structure shown in Formula I and a second chain having a structure shown in Formula II,
  • the DLL3-VH has: a DLL3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a DLL3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a DLL3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 16; and the DLL3-VL has: a DLL3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 17, a DLL3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a DLL3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and
  • the CD3-VH comprises: a CD3-HCDR1 comprising an amino acid sequence of SEQ ID NO: 56, a CD3-HCDR2 comprising an amino acid sequence of SEQ ID NO: 57, and a CD3-HCDR3 comprising an amino acid sequence of SEQ ID NO: 58; and the CD3-VL comprises: a CD3-LCDR1 comprising an amino acid sequence of SEQ ID NO: 59, a CD3-LCDR2 comprising an amino acid sequence of SEQ ID NO: 60, and a CD3-LCDR3 comprising an amino acid sequence of SEQ ID NO: 61;
  • the structures shown in Formula I and Formula II are arranged from the N-terminus to the C-terminus;
  • the linker 1, linker 2 and linker 3 are identical or different peptide linkers;
  • the Fc1 and the Fc2 are Fc region structural sequences that can associate with each other using a knob-and-hole technique;
  • the buffer is a histidine buffer, an acetate buffer, a citrate buffer, a succinate buffer or a phosphate buffer.
  • the aforementioned buffer is histidine-histidine hydrochloride buffer, histidine-histidine acetate buffer or citric acid-sodium citrate buffer.
  • the buffer as described in any of the preceding items is histidine-histidine hydrochloride buffer.
  • the pharmaceutical composition as described in any of the preceding items wherein the pH of the pharmaceutical composition is 4.5 to 6.0; preferably, the pH of the pharmaceutical composition is 4.5 to 5.5; more preferably, the pH of the pharmaceutical composition is 4.8 to 5.2. In some embodiments, the pH of the pharmaceutical composition is about 4.5. In some embodiments, the pH of the pharmaceutical composition is 4.5. In some embodiments, the pH of the pharmaceutical composition is about 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.0.
  • the pH of any of the pharmaceutical compositions described is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, or any range between these point values.
  • a point value is mentioned in this disclosure, it should be understood that the point value includes a margin of error. This margin of error is due to factors such as laboratory environment, personnel operation, instrumentation, methodology, and measurement error. Taking pH as an example, when the measured value is about 5.0, it should be understood that it includes a margin of error.
  • “about 5.0” means 5.0 ⁇ 0.2 (i.e., pH is 4.8 to 5.2).
  • the pharmaceutical composition as described in any of the preceding items wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg/mL to 150 mg/mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 5 mg/mL to 150 mg/mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg/mL to 120 mg/mL.
  • the pharmaceutical composition as described in any of the preceding items wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg/mL to 100 mg/mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 5 mg/mL to 100 mg/mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 70 mg/mL to 70 mg/mL.
  • the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 1 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 1 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 5 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 5 mg/mL.
  • the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 70 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 70 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is about 100 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds to DLL3 and CD3 is 100 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds DLL3 and CD3 is about 150 mg/mL. In some embodiments, the pharmaceutical composition, wherein the concentration of the bispecific antibody that specifically binds DLL3 and CD3 is 150 mg/mL.
  • the pharmaceutical composition of any of the preceding items, wherein the concentration of the bispecific antibody that specifically binds DLL3 and CD3 is 1 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 10 ...
  • 0 mg/mL 95 mg/mL, 100 mg/mL, 105 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, 200 mg/mL, 210 mg/mL, 220 mg/mL, 230 mg/mL, 240 mg/mL or 250 mg/mL, or any range between these points.
  • the pharmaceutical composition wherein the concentration of the bispecific antibody that specifically binds DLL3 and CD3 is about 1 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL.
  • the present invention relates to an aqueous solution of at least about 200 mg/mL of sodium bicarbonate, about 210 mg/mL, about 220 mg/mL, about 230 mg/mL, about 240 mg/mL, or about 250 mg/mL.
  • the surfactant is a nonionic surfactant.
  • the surfactant is selected from poloxamer (e.g., P188), polysorbate (e.g., polysorbate 20, polysorbate 80), Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl -Betaine, cetyl-betaine, lauroyl prop
  • the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80 or poloxamer 188 (P188). In some embodiments, the surfactant is polysorbate 80.
  • a pharmaceutical composition as described in any of the preceding items wherein the concentration of polysorbate 80 is 0.01 mg/mL to 1.0 mg/mL. In some embodiments, the concentration of polysorbate 80 is 0.1 mg/mL to 1.0 mg/mL. In some embodiments, the concentration of polysorbate 80 is 0.2 mg/mL to 0.6 mg/mL. In some embodiments, the concentration of polysorbate 80 is 0.3 mg/mL to 0.5 mg/mL. In some embodiments, the concentration of polysorbate 80 is about 0.1 mg/mL. In some embodiments, the concentration of polysorbate 80 is 0.1 mg/mL. In some embodiments, the concentration of polysorbate 80 is about 0.4 mg/mL.
  • the concentration of polysorbate 80 is 0.4 mg/mL. In some embodiments, the concentration of polysorbate 80 is about 1.0 mg/mL. In some embodiments, the concentration of polysorbate 80 is 1.0 mg/mL. In some embodiments, the concentration of polysorbate 80 is 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.15 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL or 1.0 mg/mL, or any range between these points.
  • the polysorbate 80 concentration is about 0.01 mg/mL, about 0.05 mg/mL, about 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, or about 1.0 mg/mL.
  • a pharmaceutical composition as described in any of the preceding items wherein the concentration of poloxamer 188 is 0.5 mg/mL to 5 mg/mL. In some embodiments, the concentration of poloxamer 188 is 1 mg/mL to 3 mg/mL. In some embodiments, the concentration of poloxamer 188 is 1.5 mg/mL to 2.5 mg/mL. In some embodiments, the concentration of poloxamer 188 is 1.8 mg/mL to 2.2 mg/mL. In some embodiments, the concentration of poloxamer 188 is about 2 mg/mL. In some embodiments, the concentration of poloxamer 188 is 2 mg/mL.
  • the poloxamer 188 concentration is 0.5 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2 mg/mL, 2.1 mg/mL, 2.2 mg/mL, 2.3 mg/mL, 2.4 mg/mL, 2.5 mg/mL, 2.6 mg/mL, 2.8 mg/mL, 3 mg/mL, 3.5 mg/mL, 4 mg/mL, 4.5 mg/mL or 5.0 mg/mL, or any range between these points.
  • the poloxamer 188 concentration is about 0.5 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 1.6 mg/mL, about 1.7 mg/mL, about 1.8 mg/mL, about 1.9 mg/mL, about 2 mg/mL, about 2.1 mg/mL, about 2.2 mg/mL, about 2.3 mg/mL, about 2.4 mg/mL, about 2.5 mg/mL, about 2.6 mg/mL, about 2.8 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, or about 5.0 mg/mL.
  • the pharmaceutical composition as described in any of the preceding items comprises sugar.
  • the sugar is selected from the conventional composition ( CH2O ) n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.
  • the sugar can be selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibitose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.
  • the sugar as described above is sucrose, trehalose, mannitol or sorbitol. In some embodiments, the sugar is sucrose.
  • a pharmaceutical composition as described in any of the preceding items wherein the concentration of the sugar is 10 mg/mL to 120 mg/mL. In some embodiments, the concentration of the sugar is 30 mg/mL to 100 mg/mL. In some embodiments, the concentration of the sugar is 64 mg/mL to 96 mg/mL. In some embodiments, the concentration of the sugar is 68 mg/mL to 92 mg/mL. In some embodiments, the concentration of the sugar is 72 mg/mL to 88 mg/mL. In some embodiments, the concentration of the sugar is about 30 mg/mL. In some embodiments, the concentration of the sugar is about 80 mg/mL. In some embodiments, the concentration of the sugar is about 100 mg/mL.
  • the concentration of the sugar is non-limiting and includes 10mg/mL, 20mg/mL, 30mg/mL, 35mg/mL, 37.5mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 55mg/mL, 60mg/mL, 64mg/mL, 65mg/mL, 68mg/mL, 70mg/mL, 72mg/mL, 75mg/mL, 80mg/mL, 85mg/mL, 88mg/mL, 90mg/mL, 92mg/mL, 95mg/mL, 96mg/mL, 100mg/mL or 120mg/mL, and any range between these point values.
  • the concentration of the sugar is 30mg/mL. In some embodiments, the concentration of the sugar is 80mg/mL. In some embodiments, the concentration of the sugar is 100mg/mL. In some embodiments, the concentration of the sugar includes, but is not limited to, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 35 mg/mL, about 37.5 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 64 mg/mL, about 65 mg/mL, about 68 mg/mL, about 70 mg/mL, about 72 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 88 mg/mL, about 90 mg/mL, about 92 mg/mL, about 95 mg/mL, about 96 mg/mL, about 100 mg/mL, or about 120 mg/mL.
  • a pharmaceutical composition as described in any of the preceding items wherein the concentration of the sucrose is 10 mg/mL to 120 mg/mL. In some embodiments, the concentration of the sucrose is 30 mg/mL to 100 mg/mL. In some embodiments, the concentration of the sucrose is 64 mg/mL to 96 mg/mL. In some embodiments, the concentration of the sucrose is 68 mg/mL to 92 mg/mL. In some embodiments, the concentration of the sucrose is 72 mg/mL to 88 mg/mL. In some embodiments, the concentration of the sucrose is about 30 mg/mL. In some embodiments, the concentration of the sucrose is about 80 mg/mL.
  • the concentration of the sucrose is about 100 mg/mL. In some embodiments, the concentration of the sucrose is non-limiting and includes 10mg/mL, 20mg/mL, 30mg/mL, 35mg/mL, 37.5mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 55mg/mL, 60mg/mL, 64mg/mL, 65mg/mL, 68mg/mL, 70mg/mL, 72mg/mL, 75mg/mL, 80mg/mL, 85mg/mL, 88mg/mL, 90mg/mL, 92mg/mL, 95mg/mL, 96mg/mL, 100mg/mL or 120mg/mL, and any scope between these point values.
  • the concentration of the sucrose is 30mg/mL. In some embodiments, the concentration of the sucrose is 80mg/mL. In some embodiments, the concentration of the sucrose is 100mg/mL. In some embodiments, the concentration of sucrose includes, but is not limited to, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 35 mg/mL, about 37.5 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 64 mg/mL, about 65 mg/mL, about 68 mg/mL, about 70 mg/mL, about 72 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 88 mg/mL, about 90 mg/mL, about 92 mg/mL, about 95 mg/mL, about 96 mg/mL, about 100 mg/mL, or about 120 mg/mL
  • a pharmaceutical composition as described in any of the preceding items wherein the concentration of the buffer is 5mM to 100mM. In some embodiments, the concentration of the buffer is 10mM to 50mM. In some embodiments, the concentration of the buffer is 10mM to 30mM. In some embodiments, the concentration of the buffer is 15mM to 25mM. In some embodiments, the concentration of the buffer is 24mM to 36mM. In some embodiments, the concentration of the buffer is 27mM to 33mM. In some embodiments, the concentration of the buffer is 14mM to 22mM. In some embodiments, the concentration of the buffer is 16mM to 20mM. In some embodiments, the concentration of the buffer is about 30mM.
  • the concentration of the buffer is 18mM. In some embodiments, the concentration of the buffer is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 33 mM, about 36 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.
  • the concentration of the histidine-histidine hydrochloride buffer is 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 27mM, 30mM, 33mM, 36mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, and any range between these point values. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 30 mM.
  • the concentration of the excipient is 0.09 mg/mL to 0.11 mg/mL. In some embodiments, the concentration of the excipient is about 0.01 mg/mL. In some embodiments, the concentration of the excipient is about 0.1 mg/mL. In some embodiments, the concentration of the excipient is about 1 mg/mL.
  • the concentration of the excipient is 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.11 mg/mL, 0.12 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.15 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL or 1 mg/mL, and any range between these point values.
  • the pharmaceutical composition as described in any of the preceding items wherein the concentration of the excipient is 0.01 mg/mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the excipient is 0.1 mg/mL. In some embodiments, the pharmaceutical composition as described in any of the preceding items, wherein the concentration of the excipient is 1 mg/mL.
  • the concentration of the excipient is about 0.01 mg/mL, about 0.03 mg/mL, about 0.05 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.11 mg/mL, about 0.12 mg/mL, about 0.13 mg/mL, about 0.14 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, or about 1 mg/mL.
  • the pharmaceutical composition as described in any of the preceding items wherein the excipient is 0.01mg/mL to 1mg/mL of disodium ethylenediaminetetraacetate dihydrate. In some embodiments, the excipient is 0.01mg/mL to 0.5mg/mL of disodium ethylenediaminetetraacetate dihydrate. In some embodiments, the excipient is 0.01mg/mL to 0.2mg/mL of disodium ethylenediaminetetraacetate dihydrate. In some embodiments, the excipient is 0.01mg/mL to 0.12mg/mL of disodium ethylenediaminetetraacetate dihydrate.
  • the concentration of disodium ethylenediaminetetraacetate is about 0.01 mg/mL, about 0.03 mg/mL, about 0.05 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.11 mg/mL, about 0.12 mg/mL, about 0.13 mg/mL, about 0.14 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, or about 1 mg/mL.
  • the DLL3-VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the DLL3-VL comprises the amino acid sequence shown in SEQ ID NO: 42; and
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 62
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 63.
  • amino acid sequence of the DLL3-VH is shown in SEQ ID NO: 33, and the amino acid sequence of the DLL3-VL is shown in SEQ ID NO: 42; and
  • the amino acid sequence of the CD3-VH is shown in SEQ ID NO: 62
  • the amino acid sequence of the CD3-VL is shown in SEQ ID NO: 63.
  • the pharmaceutical composition as described in any of the preceding items wherein the bispecific antibody specifically binds to DLL3 and CD3, wherein the Fc1 has a protrusion structure according to the knob-in-hole technique, and the Fc2 has a hole structure according to the knob-in-hole technique.
  • the amino acid sequence of the Fc1 in the bispecific antibody that specifically binds DLL3 and CD3 is as shown in SEQ ID NO: 64; and the amino acid sequence of the Fc2 is as shown in SEQ ID NO: 65.
  • the amino acid sequence of the Fc1 in the bispecific antibody that specifically binds DLL3 and CD3 is as shown in SEQ ID NO: 66; and the amino acid sequence of the Fc2 is as shown in SEQ ID NO: 67.
  • the linker 1, linker 2, and linker 3 are all peptide linkers known in the art, as long as the bispecific antibody can exhibit the desired antigen binding activity.
  • the peptide linker can be a flexible peptide comprising 1-50 or 3-20 amino acid residues.
  • the length of the peptide linker is 1-15 amino acid residues.
  • the linker 1 has a structure as shown in the general sequence formula (GGGS)nGm, wherein n is 1-5, preferably 1, 2 or 3; m is 1-10, preferably 4, 5, 6, 7 or 8.
  • the sequence of the linker 1 is GGGSGGGG (SEQ ID NO: 68).
  • the linker 2 has a structure as shown in the general sequence formula Gm, wherein m is 1-10, preferably 1-5, more preferably 1, 2 or 3.
  • the sequence of the linker 2 is G (SEQ ID NO: 69).
  • the linker 3 has a structure as shown in the sequence formula (GGGGS)nGm, wherein n is 1-5, preferably 1, 2 or 3; m is 1-10, preferably 4, 5, 6, 7 or 8.
  • the sequence of linker 3 is GGGGSGGGG (SEQ ID NO: 70).
  • Linker 1, Linker 2, and Linker 3 in the bispecific antibody that specifically binds to DLL3 and CD3 are the same or different, and the amino acid sequences are each independently selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.
  • the amino acid sequences of Linker 1, Linker 2 and Linker 3 in the bispecific antibody that specifically binds to DLL3 and CD3 are SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, respectively.
  • amino acid sequence of Formula I i.e., chain 1
  • amino acid sequence of Formula II i.e., chain 2
  • SEQ ID NO: 72 amino acid sequence of Formula II
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pH of the pharmaceutical composition is 4.5 to 6.0.
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pH of the pharmaceutical composition is 4.5 to 6.0.
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the pharmaceutical composition as described in any of the preceding items comprises the following components:
  • the present disclosure provides a method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition as described in any of the preceding items.
  • the present disclosure provides a lyophilized formulation obtained by the method as described above.
  • the pharmaceutical composition, lyophilized formulation or reconstituted solution as described in any of the preceding items is suitable for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation or reconstituted solution as described in any of the preceding items is suitable for subcutaneous injection.
  • the pharmaceutical composition, lyophilized preparation or reconstituted solution as described in any of the preceding items is used to prepare a drug for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized preparation or reconstituted solution as described in any of the preceding items is used to prepare a drug for subcutaneous injection.
  • the present disclosure also provides a pharmaceutical composition as described in any of the preceding items for use as a medicament, or a lyophilized formulation as described in any of the preceding items, or an injection formulation as described in any of the preceding items.
  • the medicament is used to treat a tumor or cancer.
  • the present disclosure also provides a method for treating tumors or cancers, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition as described in any of the preceding items, or the lyophilized preparation as described in any of the preceding items, or the injectable preparation as described in any of the preceding items.
  • the present disclosure provides use of the composition as described in any of the preceding items, or the lyophilized preparation as described in any of the preceding items, or the injectable preparation as described in any of the preceding items in the preparation of a medicament for treating a tumor or cancer.
  • the tumor or cancer described in any of the preceding items is selected from the group consisting of lung cancer, small cell lung cancer, large cell lung cancer, head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, large cell lung cancer, triple negative breast cancer and lymphom
  • the tumor or cancer as described in any of the preceding items is a solid tumor.
  • the tumor or cancer as described in any of the preceding items is lung cancer.
  • the tumor or cancer as described in any of the preceding items is small cell lung cancer.
  • the method for treating a tumor or cancer as described in any of the preceding items further comprises the use of a second therapeutic agent.
  • the second therapeutic agent comprises an anti-tumor agent, radiotherapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an anti-tumor agent, an immune checkpoint inhibitor, or a combination thereof.
  • the second therapeutic agent in the method for treating a tumor or cancer as described above, is administered simultaneously, sequentially, or separately with the bispecific antibody formulation described in any one of the present disclosures.
  • the bispecific antibody preparation provided by the present disclosure has the characteristics of good therapeutic activity, safety, pharmacokinetic properties and drugability (such as stability).
  • Figure 2A shows the experimental results of the binding of bispecific antibodies to DLL1. The results show that the DLL3-CD3 bispecific antibody disclosed herein does not bind to DLL1.
  • Figure 2B shows the experimental results of the binding of bispecific antibodies to DLL4. The results show that the DLL3-CD3 bispecific antibodies disclosed herein do not bind to DLL4.
  • FIG4A shows the experimental results of the activation of T cells by bispecific antibodies, and the results show that the bispecific antibodies disclosed herein have a significant effect of activating T cells in the presence of DLL3/H82 cells expressing DLL3.
  • FIG. 4B shows that the bispecific antibodies of the present disclosure do not activate T cells in the presence of negative cells H460 that do not express DLL3.
  • FIG5A shows the experimental results of cytokine release of the bispecific antibody, and the results show that in the presence of H1184 cells, the bispecific antibody of the present disclosure stimulates PBMC cells to secrete very low levels of IFN ⁇ .
  • FIG5B shows that in the presence of H1184 cells, the bispecific antibodies of the present disclosure stimulated PBMC cells to secrete very low levels of IL-6.
  • DLL3 Unless specified as being from a non-human species (e.g., “mouse DLL3,” “mouse DLL3 fragment,” “monkey DLL3,” “monkey DLL3 fragment,” etc.), “DLL3” and “DLL3 fragment” as used herein refer to the well-known human DLL3 protein or a fragment thereof.
  • CD3 refers to the well-known human CD3 protein or a fragment thereof.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.
  • Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.
  • amino acid mutation includes amino acid substitution, deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, as long as the final construct has the desired properties, such as reduced or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain.
  • Specific amino acid mutations can be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, that is, an amino acid is replaced with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include replacement by non-natural amino acids or by derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is expected that methods other than genetic engineering to change amino acid side chain groups, such as chemical modifications, are also available. Various names can be used herein to indicate the same amino acid mutation.
  • the amino acid residue at a specific position can be represented by position+amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has mutated from T to W.
  • antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antibody fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Depending on the context, the skilled person can determine the specific meaning of "antibody”.
  • “Native antibody” refers to naturally occurring immunoglobulin molecules.
  • a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two light chains and two heavy chains bound by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH, also known as variable heavy domain, heavy chain variable region), followed by three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL, also known as variable light domain, or light chain variable domain), followed by a constant light domain (light chain constant region, CL).
  • VH variable heavy domain
  • CH1 variable heavy domain
  • CL constant light domain
  • full-length antibody complete antibody
  • whole antibody are used interchangeably herein and refer to an antibody having a structure substantially similar to a natural antibody structure or having a heavy chain containing an Fc region as defined herein.
  • bispecific antibody refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen.
  • the prior art has disclosed bispecific antibodies of various structures, which can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies according to the integrity of the IgG molecule; bispecific antibodies of divalent, trivalent, tetravalent or more valence according to the number of antigen-binding regions; and bispecific antibodies of symmetrical structure and asymmetrical structure according to whether the structure is symmetrical.
  • bispecific antibodies based on antibody fragments are formed by combining two or more Fab fragments into one molecule. They have low immunogenicity, small molecular weight, and high tumor tissue permeability.
  • Typical antibody structures of this type include bispecific antibodies such as F(ab)2, scFv-Fab, (scFv)2-Fab, etc.; IgG-like bispecific antibodies (for example, with Fc fragments) have a relatively large molecular weight.
  • the Fc fragment helps in the later purification of antibodies and improves their solubility and stability.
  • the Fc part may also bind to the receptor FcRn to increase the serum half-life of the antibody.
  • Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, and XmA b, 2: 1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al.,
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia” numbering rule, “ABM” numbering rule, "contact” numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering rule (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art.
  • the numbering convention for this disclosure is shown in Table 1 below.
  • variable region and CDR sequences in the disclosed embodiments are all subject to the "Kabat" numbering convention.
  • antibody fragment refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that retains the antigen-binding ability of the intact antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • Fc region or “fragment crystallizable region” is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and modified Fc regions.
  • the Fc region comprises two identical or different subunits.
  • the Fc region of the human IgG heavy chain is defined as extending from the amino acid residue at the Cys226 position or from Pro230 to its carboxyl terminus.
  • Suitable native sequence Fc regions for antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise indicated, the numbering convention for the Fc region is the EU index.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the constant region and the framework region portion of the variable region).
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen).
  • KD equilibrium dissociation constant
  • KD refers to an equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as a molar concentration (M).
  • M molar concentration
  • polyclonal antibody refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for possible natural mutations that are present in small amounts.
  • polyclonal antibody preparations typically contain a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes.
  • the antibodies provided by the present disclosure are monoclonal antibodies.
  • antigen refers to a molecule or portion of a molecule that can be bound by a selective binding agent of an antigen binding molecule (eg, an antibody).
  • An antigen may have one or more epitopes that can interact with different antigen binding molecules (eg, antibodies).
  • epitope refers to an area or region on an antigen that is capable of specific binding to an antibody or its antigen-binding fragment.
  • An epitope may be formed by contiguous amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, non-contiguous amino acids being brought into spatial proximity by the folding of the antigen (i.e., by tertiary folding of the antigen of a protein nature).
  • Conformational epitopes differ from linear epitopes in that in the presence of denaturing solvents, antibody binding to the conformational epitope is lost.
  • An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind to a specific epitope can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).
  • an antigen binding molecule is capable of binding to a certain antigen or epitope with a higher affinity than other antigens or epitopes.
  • an antibody binds to an antigen or epitope with an equilibrium dissociation constant (KD) of about 1 ⁇ 10-7 M or less (e.g., about 1 ⁇ 10-8 M or less).
  • KD equilibrium dissociation constant
  • the KD of an antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein).
  • KD can be measured using known methods, such as those measured by FACS or surface plasmon resonance assays.
  • an antibody that specifically binds to an antigen or an epitope thereof may have cross-reactivity to other related antigens, for example, cross-reactivity to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes (chimpanzee, chimp), or marmoset (Callithrix jacchus (common marmoset, marmoset).
  • homologous such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes (chimpanzee, chimp), or marmoset (Callithrix jacchus (common marmoset, marmoset).
  • does not bind means that the antigen-binding molecule is unable to bind to an antigen or an epitope thereof in the above-mentioned specific binding manner.
  • KD equilibrium dissociation constant
  • antigen binding moiety refers to a polypeptide molecule that specifically binds to a target antigen.
  • Specific antigen binding moieties include the antigen binding domain of an antibody, for example, comprising a heavy chain variable region and a light chain variable region.
  • antigen binding moiety that specifically binds to CD3 refers to a module that can bind to CD3 or its epitope with sufficient affinity so that a molecule containing the module can be used as a diagnostic and/or therapeutic agent targeting CD3.
  • an antigen binding moiety that specifically binds to CD3 has the following equilibrium dissociation constant (KD): ⁇ about 10 nM, which is measured by surface plasmon resonance assay.
  • Antigen binding moieties include antibody fragments as defined herein, such as Fab, replaced Fab or scFv.
  • linker refers to a connecting unit that connects two polypeptide fragments.
  • the linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids.
  • the linkers used in this article can be the same or different.
  • Tm is the melting denaturation temperature (intrinsic fluorescence). When a protein denatures (heat or denaturant), the tertiary structure opens, the aromatic amino acid microenvironment changes, and the emission fluorescence spectrum changes.
  • Tm1 refers to the temperature at which the fluorescence changes to half of the maximum value.
  • Tonset is the denaturation starting temperature, which refers to the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.
  • Tagg is the aggregation onset temperature. Aggregates are detected at two wavelengths, 266nm and 473nm, by static light scattering, and the temperature at which the sample begins to aggregate is monitored. Tagg 266 refers to the aggregation onset temperature monitored at 266nm.
  • fused or "linked” means that the components (eg, an antigen binding moiety and an Fc domain) are covalently linked directly or via a linker.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide connected thereto.
  • plasmid refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected.
  • viral vector such as an adeno-associated virus vector (AAV or AAV2), wherein additional DNA segments can be connected to the viral genome.
  • AAV adeno-associated virus vector
  • Certain vectors can replicate autonomously in the host cell in which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins).
  • vectors can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome.
  • expression vector or "expression construct” refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence for guiding and/or controlling (together with the host cell) the expression of one or more heterologous coding regions operably connected thereto.
  • the expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and affect RNA splicing of the coding region operably connected thereto when introns are present.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein.
  • cyno or “cynomolgus” refers to cynomolgus monkeys (Macaca fascicularis).
  • the individual or subject is a human.
  • administering refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory
  • Treatment refers to clinical interventions attempted to be applied to the individual being treated, and may be performed for preventive purposes or during the course of clinical pathology.
  • the desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and regression or improved prognosis.
  • recurrence refers to the return of cancer or disease after clinical assessment of disease resolution.
  • diagnosis of distant metastasis or local recurrence may be considered a recurrence.
  • an "effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or potential causes, prevent the occurrence of symptoms and/or their potential causes, and/or improve or ameliorate damage caused by or associated with the disease state.
  • an effective amount is a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” is an amount sufficient to treat a disease state or symptoms, particularly a state or symptoms associated with the disease state, or otherwise prevent, hinder, delay or reverse the progression of the disease state or any other undesirable symptoms in any way associated with the disease.
  • a “prophylactically effective amount” is an amount that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms.
  • a complete therapeutic or preventive effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, a therapeutic or preventive effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “prophylactically effective amount” may vary depending on a variety of factors: such as the disease state, age, sex, and weight of an individual, and the ability of a therapeutic agent or combination of therapeutic agents to elicit a desired response in an individual.
  • Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health conditions of the subject.
  • Buffer refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components.
  • buffers that control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.
  • Hetidine buffer is a buffer containing histidine.
  • histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, histidine-histidine sulfate and the like, preferably histidine-histidine hydrochloride buffer.
  • Histidine-histidine hydrochloride buffer can be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.
  • citrate buffer is a buffer including citrate ions.
  • citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer is citric acid-sodium citrate.
  • succinate buffer is a buffer comprising succinate ions.
  • succinate buffers include succinic acid-succinic acid sodium salt, succinic acid-succinic acid potassium salt, succinic acid-succinic acid calcium salt, etc.
  • a preferred succinate buffer is succinic acid-succinic acid sodium salt.
  • the succinic acid-succinic acid sodium salt can be prepared from succinic acid and sodium hydroxide, or from succinic acid and succinic acid sodium salt.
  • Phosphate buffer is a buffer including phosphate ions.
  • phosphate buffers include citric acid-disodium hydrogen phosphate, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like.
  • a preferred phosphate buffer is citric acid-disodium hydrogen phosphate.
  • Acetate buffer is a buffer including acetate ions.
  • acetate buffers include acetate-sodium acetate, histidine-histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, etc.
  • a preferred acetate buffer is acetate-sodium acetate.
  • Polyxamer is an ⁇ -hydrogen- ⁇ -hydroxy poly(ethylene oxide) a-poly(propylene oxide) b-poly(ethylene oxide) a block copolymer. Propylene oxide and propylene glycol react to form polyoxypropylene glycol, and then ethylene oxide is added to form a block copolymer. Wherein, a is the number of ethylene oxide units, and b is the number of propylene oxide units. Examples of poloxamers include, but are not limited to, poloxamer 188 (P188 or PF68).
  • poloxamer 188 in the copolymer, the ethylene oxide units (a) are 75 to 85, the propylene oxide units (b) are 25 to 30, the ethylene oxide (EO) content is 79.9% to 83.7%, and the average molecular weight is 7680 to 9510.
  • “Lyophilized preparation” refers to a pharmaceutical composition in liquid or solution form or a preparation or pharmaceutical composition obtained after a liquid or solution preparation has been subjected to a vacuum freeze-drying step.
  • the pharmaceutical composition disclosed herein can achieve a stable effect: the antibody therein substantially retains its physical stability and/or chemical stability and/or biological activity after storage.
  • the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage.
  • the storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition.
  • analytical techniques for measuring protein stability which can measure stability after storage at a selected temperature for a selected period of time.
  • a stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 1 month, at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years.
  • a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after being stored at a temperature of 25°C for a period of 1 month, 3 months, or 6 months. It also includes a temperature of 40°C that exhibits desired characteristics after being stored for a period of 4 weeks, 1 month, 3 months, or 6 months.
  • a typical example of stability measured by SEC-HPLC, usually no more than about 10%, preferably no more than about 5%, of the antibody aggregates or degrades.
  • the formulation is a light yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly milky white.
  • concentration, pH, weight, and molecular osmotic pressure of the formulation have no more than ⁇ 10% variation, preferably no more than ⁇ 5% variation.
  • the formulation generally forms no more than about 10%, preferably no more than about 5% aggregation.
  • An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and/or denaturation as measured after visual inspection of color and/or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines protein tertiary structure, and by FTIR spectroscopy, which determines protein secondary structure.
  • An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.
  • antigen binding molecule variants with one or more amino acid substitutions are provided. Substitutions can be made in CDR and FR. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions”. More substantial changes are provided in Table 2 under the heading of "exemplary substitutions", and are further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product is screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions would involve exchanging a member from one class for a member from another class.
  • a class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., humanized or human antibody).
  • a parent antibody e.g., humanized or human antibody
  • the resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will substantially retain certain biological properties of the parent antibody.
  • An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced, for example, using affinity maturation techniques based on phage display (such as those described herein).
  • affinity maturation techniques based on phage display (such as those described herein).
  • one or more CDR residues are mutated, and the variant antibody is displayed on phage, and a specific biological activity (e.g., binding affinity) is screened for it.
  • Changes can be made to CDR, for example, to improve antibody affinity.
  • Such changes can be made to CDR "hot spots", i.e., residues encoded by codons that undergo mutations at high frequencies during the somatic maturation process, and/or residues that contact antigens, while the resulting variant VH or VL is tested for binding affinity.
  • affinity maturation diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-guided mutagenesis). Then, a secondary library is created. Then, the library is screened to identify any antibody variant with the desired affinity.
  • Another method of introducing diversity involves a CDR-directed method in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues that are involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen.
  • conservative changes e.g., conservative substitutions, as provided herein
  • each CDR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.
  • a method that can be used to identify residues or regions in an antibody that can be used as a target for mutagenesis is called "alanine scanning mutagenesis".
  • a residue or group of residues e.g., charged residues, such as Arg, Asp, His, Lys, and Glu
  • neutral or negatively charged amino acids e.g., Ala or polyalanine
  • Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution.
  • the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain desired properties.
  • Amino acid sequence insertions include: fusions of 1 residue or polypeptides of 100 or more residues in length at the amino and/or carboxyl termini; and intrasequence insertions of single or multiple amino acid residues.
  • Examples of insertions at the terminals include antibodies with an N-terminal methionyl residue.
  • Other insertion variants of antibody molecules include fusions with enzymes (or polypeptides that extend the serum half-life of antibodies) fused to the N or C termini of the antibody.
  • the Fc region of the bispecific antibody of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, such as its binding to an Fc ⁇ receptor, and reduce or eliminate effector function.
  • a native IgG Fc region specifically an IgG 1 Fc region or an IgG 4 Fc region, may cause the bispecific antibody of the present disclosure to target cells expressing Fc receptors rather than cells expressing antigens.
  • the modified Fc region of the present disclosure exhibits reduced binding affinity to Fc receptors and/or reduced effector function.
  • the modified Fc region has a binding affinity to Fc receptors that is reduced by 50%, 80%, 90% or more than 95% compared to the native Fc region.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is a human Fc ⁇ receptor, such as Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the modified Fc region also has reduced binding affinity for complement, such as C1q, compared to the native Fc region.
  • the modified Fc region does not have reduced binding affinity for neonatal Fc receptor (FcRn) compared to the native Fc region.
  • the modified Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.
  • CDC complement dependent cytotoxicity
  • ADCC reduced antibody-dependent cell-mediated cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • reduced immune complex-mediated antigen uptake by antigen presenting cells reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptos
  • substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327 and 329, etc. can reduce effector function.
  • the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbering is according to the EU index.
  • substitution of amino acid residues at positions 228, etc. can reduce effector function.
  • the Fc region disclosed herein comprises a transformation according to the knob-into-hole (KIH) technology, which involves introducing a protrusion structure (knob) at the interface of the first subunit and introducing a hole structure (hole) at the interface of the second subunit.
  • the protrusion structure is positioned in the hole structure, promoting the formation of heterodimers and inhibiting the generation of homodimers.
  • the protrusion structure is constructed by replacing the small amino acid side chains from the interface of the first subunit with larger side chains (such as tyrosine or tryptophan).
  • the hole structure is created in the interface of the second subunit by replacing the large amino acid side chains with smaller amino acid side chains (such as alanine or threonine).
  • the protrusion structure and the hole structure are prepared by changing the nucleic acid encoding the polypeptide, and the optional amino acid substitutions are shown in the following table:
  • knob-in-hole technology In addition to the knob-in-hole technology, other technologies for modifying the CH3 domain of the heavy chain to achieve heterodimerization are also known in the art, such as WO1996027011A1, WO1998050431, EP1870459, WO2007110205, WO2009089004, WO2010129304, WO201190754, WO2011143545, WO2012058768, WO2013157954 and WO2013096291.
  • nucleic acids are required, for example, one for the first light chain, one for the first heavy chain comprising a first heterologous monomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide.
  • These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, i.e. one vector may contain more than one of these nucleic acids.
  • the disclosure provides an isolated nucleic acid encoding an antibody as described above. Such nucleic acids can independently encode any of the aforementioned polypeptide chains.
  • the disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids.
  • the disclosure provides a host cell comprising such nucleic acids.
  • a method for preparing a bispecific antibody comprises, under conditions suitable for antibody expression, culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, and optionally recovering the antibody from a host cell (or host cell culture medium).
  • the nucleic acid encoding the protein is separated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • Such nucleic acids can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody), or produced by recombinant methods or obtained by chemical synthesis.
  • Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required for the antibodies. After expression, the antibodies can be separated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized” to produce antibodies with partially or fully human glycosylation patterns.
  • Suitable host cells for expressing (glycosylated) antibodies can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells.
  • a number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, for example, US5959177, US6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted to growth in suspension.
  • Suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NS0 and Sp2/0.
  • CHO Chinese hamster ovary
  • myeloma cell lines such as Y0, NS0 and Sp2/0.
  • the pCDH lentiviral expression vector plasmid containing SEQ ID NO: 1-2 (synthesized by GENEWIZ), pCDH plasmid and pVSVG, pCMV lentiviral packaging vector were transfected into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent, and the culture supernatant containing the virus was collected, filtered and subjected to ultra-high-speed centrifugation. After discarding the supernatant, it was resuspended in 0.2 mL 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, and selected with puromycin for two to three weeks, and then single-cell sorting was performed by FACS. The selected monoclonal cell lines were expanded and frozen for subsequent experiments.
  • the pCDH-CMV-MCS-EF1-puro lentiviral expression vector plasmid (synthesized by GENEWIZ) containing LUC and GFP genes was transfected into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent.
  • the culture supernatant containing the virus was collected, filtered and subjected to ultra-high-speed centrifugation. After discarding the supernatant, it was resuspended in 0.2 mL of sterile PBS.
  • the concentrated virus was used to infect SHP77 (ATCC, CRL-2195), H1184 (ATCC, CRL-5858), and H460 (Chinese Academy of Sciences Cell Bank, TCHu205) respectively. After puromycin selection for two to three weeks, single-cell sorting was performed by FACS. The selected monoclonal cell lines were expanded and frozen for subsequent experiments.
  • Cynomolgus monkey DLL3 full-length protein (SEQ ID NO: 2):
  • Rat DLL3 full-length protein (SEQ ID NO: 3):
  • Mouse DLL3 full-length protein (SEQ ID NO: 4):
  • Human DLL1 full-length protein (SEQ ID NO: 5):
  • Human DLL3 (Uniprot, Q9NYJ7), cynomolgus monkey DLL3 (Uniprot, A0A2K5WSR4) and mouse DLL3 (Uniprot, O88516) sequences were used as templates to design human DLL3 ECD fusion proteins containing different tags, which were cloned into pTT5 vectors and expressed in 293E cells to obtain antigens.
  • the amino acid sequences of the relevant proteins are as follows:
  • the dot-dashed line represents the signal peptide sequence
  • the single dash line represents the his tag and linker
  • the double dash line represents the DLL3 extracellular region.
  • the dot-dashed line represents the signal peptide sequence
  • the single dash line represents the Fc tag and linker
  • the double dash line represents the DLL3 extracellular region.
  • the dot-dashed line represents the signal peptide sequence
  • the double-dashed line represents the DLL3 extracellular region
  • the single-dashed line represents the strep twin tag.
  • the dot-dashed line represents the signal peptide sequence
  • the double-dashed line represents the DLL3 extracellular region
  • the single-dashed line represents the strep twin tag.
  • the dot-dashed line represents the signal peptide sequence
  • the double-dashed line represents the DLL3 extracellular region
  • the single-dashed line represents the strep twin tag.
  • Monoclonal antibodies against human DLL3 were prepared by hybridoma technology, and the immunization method was as follows:
  • the first group used His-hDLL3 (ECD) (SEQ ID NO: 7) as the immunogen.
  • Gold Adjuvant Sigma Cat No.T2684
  • Thermo Alum Thermo Cat No.77161
  • Antigen and adjuvant ( Gold Adjuvant) ratio was 1:1
  • antigen and adjuvant (Thermo The ratio of 1:1 was 3:1, 50 ⁇ g/mouse/time (first immunization), 25 ⁇ g/mouse/time (boost immunization).
  • the antibody titer in mouse serum was determined by ELISA.
  • mice were immunized with DLL3-CHO-s cells and Fc-hDLL3 (ECD) (SEQ ID NO: 8) by alternating immunization with cell and protein antigens.
  • ECD Fc-hDLL3
  • each mouse was intraperitoneally injected with 0.1 mL of saline diluted to a concentration of 10 8 /mL.
  • Gold Adjuvant Sigma Cat No.T2684
  • Thermo Alum Thermo Cat No.77161 adjuvant cross immunization.
  • Antigen and adjuvant ( Gold Adjuvant) ratio was 1:1, antigen and adjuvant (Thermo The ratio of 1:1 was 3:1, 50 ⁇ g/mouse/time (first immunization), 25 ⁇ g/mouse/time (boost immunization).
  • the antibody titer in mouse serum was determined by ELISA.
  • the spleen lymphocytes were fused with myeloma cells Sp2/0 cells ( CRL-8287 TM ) was fused to obtain hybridoma cells. According to the growth density of hybridoma cells, the hybridoma culture supernatant was detected by ELISA method binding to DLL3 protein and FACS method binding to DLL3 CHO-s cells. The clones that bind to human DLL3 protein, monkey DLL3 protein and DLL3 CHO-s cells and do not bind to wild-type CHO-s cells were selected for timely cryopreservation, amplification and one or two subcloning until a single cell clone was obtained. The hybridoma clone mAb6 was obtained through the above experimental screening.
  • the hybridoma clones were expanded and cultured, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using degenerate primers of mouse-Ig to finally obtain the variable region sequence of the antibody.
  • the heavy chain variable region and light chain variable region of the mouse antibody were cloned into the pTT5 vector plasmid containing the human IgG1 heavy chain constant region shown in SEQ ID NO:20 and the ⁇ light chain constant region shown in SEQ ID NO:21, respectively, and then transfected into HEK293 cells to obtain the anti-DLL3 chimeric antibody M6CHI.
  • the heavy and light chain variable region germline genes with high homology were selected as templates, and the CDRs of the mouse antibody were grafted into the corresponding human templates to form a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Amino acid substitutions were made to certain amino acids in the variable region, and then recombined with the constant region (exemplarily, with the human IgG1 heavy chain constant region shown in SEQ ID NO: 20 and the human ⁇ light chain constant region shown in SEQ ID NO: 21) to obtain a full-length humanized antibody.
  • the human germline light chain template of mAb6 antibody is IGKV1-16*01 or IGKV3-20*02 and IGKJ4*01, and the human germline heavy chain template is IGHV1-3*01, IGHV7-4-1*02 or IGHV3-73*01 and IGHJ6*01.
  • the sequence of the obtained hu6 humanized antibody variable region is as follows:
  • hu6L1H1 indicates that the antibody contains a heavy chain variable region hu6VH1 and a light chain variable region hu6VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 20, and the sequence of its light chain constant region is SEQ ID NO: 21, and so on.
  • the above antibodies were cloned, expressed, and purified, and humanized antibodies with better activity were finally selected through protein binding experiments (Test Example 3), cell binding experiments (Test Example 2), and Biacore (Test Example 1).
  • the heavy and light chain amino acid sequences of exemplary humanized antibodies are as follows:
  • Hu6 also known as hu6L4H12 heavy chain (SEQ ID NO: 54):
  • Hu6 also known as hu6L4H12 light chain (SEQ ID NO: 55):
  • the CD3 binding portion disclosed herein can be derived from any suitable antibody. Specifically, the embodiment disclosed herein uses S107E, whose variable region and CDR sequences are as follows:
  • variable region sequence of S107E is as follows:
  • the DLL3-CD3 bispecific antibody molecule disclosed herein comprises two chains, and the specific structure is as follows:
  • Chain 1 structure [VL (anti-DLL3)]-Linker 1-[VH (anti-CD3)]-Linker 2-[Fc (Knob, L234A, L235A, G237A, Y349C, T366W)];
  • Chain 2 structure [VL(anti-CD3)]-Linker 3-[VH(anti-DLL3)]-Linker 2-[Fc(hole,L234A,L235A,G237A,S354C,T366S,L368A,Y407V)];
  • the Fc regions of the two chains of the bispecific antibody molecule are Fc regions that can associate with each other using a knob-in-hole technique.
  • the Fc region of chain 1 has a protrusion structure according to the knob-in-hole technique (such as SEQ ID NO: 64 or SEQ ID NO: 66)
  • the Fc region of chain 2 has a hole structure according to the knob-in-hole technique (such as SEQ ID NO: 65 or SEQ ID NO: 67).
  • This test example studies the killing activity of bispecific antibodies as T cell engagement molecules on tumor cells.
  • SHP77, H1184 and H460 stable cell lines expressing LUC-GFP were used as target cells to detect the target-specific cytotoxic activity of the bispecific antibodies disclosed herein.
  • PBMC peripheral blood mononuclear cell
  • 1640+10% FBS 1640+10% FBS
  • the above PBMC suspension and target cell suspension were mixed in equal volumes, and 100 ⁇ L was added to each well to ensure that the E:T Ratio was 10:1.
  • a PBMC ONLY group was set up, and the above PBMC suspension and 1640+10% FBS complete medium were mixed in equal volumes.
  • the antibody was diluted with 1640+10% FBS medium, with a starting concentration of 600nM (6x final concentration), 5-fold dilution, 9 dose points, and 20 ⁇ L was added to each well.
  • the treated cells were cultured in an incubator at 37°C and 5% CO 2 for 48 hours.
  • the culture plate was removed, centrifuged at 1000rpm for 3 minutes, and 50 ⁇ L of supernatant was drawn into a new 3788 plate for cytokine detection and stored at -20°C.
  • 50 ⁇ L ONE-GloTM Luciferase (Promega, E6120) was added to the culture plate, incubated at room temperature for 5 minutes, and Luminescence was detected with Vendor to calculate the killing effect of the antibody at different concentrations.
  • Graphpad Prism8.0 software was used to make a dose-effect curve based on the logarithmic concentration and signal value of the antibody to obtain the IC50 of antibody-mediated killing.
  • the wells containing only target cells and PBMC without antibody were set as 0% inhibition, and the maximum inhibition rate Imax of the antibody was calculated. The results are shown in Table 15 and Figure 3.
  • the HTRF method was used to detect the secretion of cytokine IFN ⁇ in PBMCs stimulated by DLL3/CD3 dual antibodies in the presence of H1184 cells.
  • the ELISA method was used to detect the secretion of cytokine IL-6 in PBMC stimulated by the presence of DLL3/CD3 dual antibody in H1184 cells.
  • Test Example 8 Efficacy of the bispecific antibody disclosed herein in the SHP77 subcutaneous transplant tumor model
  • the present disclosure uses severe combined immunodeficient NOG mice to be inoculated with SHP-77 cells, human PBMCs are injected intraperitoneally after inoculation, and the mice are divided into groups for drug administration after tumor formation.
  • NOG mice female, weighing about 15-17 g, were purchased from Vital River. SHP-77 cells were from ATCC. Human PBMCs were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., catalog number: ID#A10S115034.
  • mice 100 ⁇ L of SHP-77 cells (4 ⁇ 10 6 cells/mouse) were inoculated subcutaneously in the right rib of NOG mice. On the day of inoculation, cryopreserved hPBMCs were thawed. On the second day of inoculation, each mouse was intraperitoneally injected with a cell suspension of 5 ⁇ 10 6 /100 ⁇ L of hPBMCs. Eight days after inoculation, when the tumor volume reached ⁇ 120 mm 3 , the body weight, tumors that were too large and too small were removed, and the mice were randomly divided into 5 groups according to the tumor volume, with 8 mice in each group. On the same day, equimolar amounts of antibodies were given, and the dosage was shown in Table 32.
  • the corresponding equimolar amounts of antibodies were injected intraperitoneally, and the drug was administered once every 5 days, for a total of 3 times, until the 15th day.
  • the tumor volume was measured twice a week, the body weight was weighed, and the data were recorded.
  • V 1/2 ⁇ L long ⁇ L short 2
  • T/C (%) (TT 0 )/ (CC 0 ) ⁇ 100%, wherein T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • TGI (%) 100-T/C (%).
  • the present disclosure uses severe combined immunodeficient NOG mice to be inoculated with H1184 cells, human PBMCs are injected intraperitoneally after inoculation, and the mice are divided into groups for drug administration after tumor formation.
  • NOG mice female, weighing about 15-17 g, were purchased from Vital River. H1184 cells were from ATCC. Human PBMCs were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd., catalog number: ID#A10S115034.
  • H1184 cells (3.5 ⁇ 10 6 cells, containing 50% MatriGel) 200 ⁇ L/mouse were inoculated subcutaneously in the right rib of NOG mice.
  • 8 frozen hPBMCs (ID#A10S115034) were resuscitated.
  • each mouse was intraperitoneally injected with a cell suspension of 5 ⁇ 10 6 /100 ⁇ L of human PBMC.
  • the tumor volume reached ⁇ 140 mm 3
  • the body weight, tumors that were too large and too small were removed, and the mice were randomly divided into groups according to the tumor volume, with 8 mice in each group.
  • V 1/2 ⁇ L long ⁇ L short 2
  • T/C (%) (TT 0 )/ (CC 0 ) ⁇ 100%, wherein T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • TGI (%) 100-T/C (%).
  • SEC% SEC monomer content percentage
  • a monomer is the peak area of the main peak monomer in the sample, and A total is the sum of all peak areas).
  • ⁇ SEC% SEC% of the preparation before the stability experiment - SEC% of the preparation after the stability experiment.

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Abstract

提供一种包含特异性结合DLL3和CD3的双特异性抗体的药物组合物。具体而言，提供一种药物组合物，包含特异性结合DLL3和CD3的双特异性抗体和缓冲剂。

Description

一种包含特异性结合DLL3和CD3的双特异性抗体的药物组合物 技术领域
本披露属于生物技术药物制剂领域，具体涉及一种包含特异性结合DLL3和CD3的双特异性抗体的药物组合物。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
小细胞肺癌(Small Cell Lung Cancer，SCLC)属于一种比较恶性的肺癌类型，占所有肺癌病例的10％-15％。小细胞肺癌的肿瘤生长速度快，容易转移，5年生存率低于7％。小细胞肺癌的治疗药物比较局限，主要是化药，如用铂类/依托泊苷联用化疗。小细胞肺癌病人对化疗初期响应良好，但极易产生耐药和复发。近几年的免疫治疗，如PD-L1抗体和PD1抗体，对SCLC患者有一定的效果，但是有效率约为15％。目前仍然还没有开发出特异性的靶向治疗药物。
DLL3是一种抑制Notch的配体，在正常状态下，DLL3处于高尔基体上，在癌细胞中(如小细胞肺癌细胞)，DLL3会表达在细胞表面，DLL3以顺式方式结合Notch，阻碍了细胞和细胞结合以及Notch在目标细胞的内吞，从而抑制了Notch信号通路，促进肿瘤细胞的生长。DLL3主要表达于神经或者神经内分泌肿瘤，包括小细胞肺癌、大细胞神经内分泌癌、胃肠道神经内分泌瘤、小细胞膀胱癌、多形性胶质细胞瘤、转移性去势性前列腺癌、黑色素瘤等，尤其是SCLC，超过80％的SCLC有DLL3的阳性表达，而正常肺癌组织及癌旁组织中不表达。这种表达的差异性使得DLL3成为治疗SCLC的一种极具潜力的治疗靶点。
CD3是一种表达于T细胞上的同种型或异型二聚体抗原。功能性CD3由四条不同链：ε、ζ、δ及γ中的两者进行二聚体结合来形成。CD3二聚体排列包括γ/ε、δ/ε及ζ/ζ。CD3与T细胞受体复合物(TCR)结合且为T细胞活化所必需。因此，已提出使用活化T细胞的抗CD3抗体用于治疗癌症。然而，抗CD3抗体的给药可能会触发T细胞活化和相关的细胞因子释放，过度的细胞因子释放导致重度细胞因子释放综合征(CRS)，其是抗CD3抗体在临床用药中的重要挑战。
发明内容
本披露提供了一种特异性结合DLL3和CD3的双特异性抗体的药物组合物及其用途。
在一个方面，本披露提供一种药物组合物，包含特异性结合DLL3和CD3的双特异性抗体和缓冲剂。
在一个方面，本披露提供一种药物组合物，包含特异性结合DLL3和CD3的双特异性抗体和缓冲剂，其中：
所述特异性结合DLL3和CD3的双特异性抗体包含一条具有式I所示结构的第一链和一条具有式II所示结构的第二链，
式I：[DLL3-VL]-[连接子1]-[CD3-VH]-[连接子2]-[Fc1]，
式II：[CD3-VL]-[连接子3]-[DLL3-VH]-[连接子2]-[Fc2]，
所述DLL3-VH具有：包含SEQ ID NO：14的氨基酸序列的DLL3-HCDR1，包含SEQ ID NO：15的氨基酸序列的DLL3-HCDR2和包含SEQ ID NO：16的氨基酸序列的DLL3-HCDR3；和所述DLL3-VL具有：包含SEQ ID NO：17的氨基酸序列的DLL3-LCDR1，包含SEQ ID NO：50的氨基酸序列的DLL3-LCDR2，和包含SEQ ID NO：19的氨基酸序列的DLL3-LCDR3；和
所述CD3-VH具有：包含SEQ ID NO：56的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：57的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：58的氨基酸序列的CD3-HCDR3；和所述CD3-VL具有：包含SEQ ID NO：59的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：60的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：61的氨基酸序列的CD3-LCDR3；
其中：式I和式II所示的结构是从N端至C端排列的；所述连接子1、连接子2和连接子3是相同或不同的肽连接子；所述Fc1和所述Fc2是能够以杵臼技术相互缔合的Fc区结构序列；
所述缓冲剂为组氨酸盐缓冲剂、醋酸盐缓冲剂、枸橼酸盐缓冲剂、琥珀酸盐缓冲剂或磷酸盐缓冲剂。
在一些实施方式中，如前所述的缓冲剂为组氨酸-盐酸组氨酸缓冲剂、组氨酸-醋酸组氨酸缓冲剂或枸橼酸-枸橼酸钠盐缓冲剂。
在一些实施方式中，如前任一项所述的所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂。
在一些实施方式中，如前任一项所述的药物组合物，其中所述药物组合物的pH为4.5至6.0；优选地，所述药物组合物的pH为4.5至5.5；更优选地，所述药物组合物的pH为4.8至5.2。在一些实施方案中，所述药物组合物的pH为约4.5。在一些实施方案中，所述药物组合物的pH为4.5。在一些实施方案中，所述药物组合物的pH为约5.5。在一些实施方案中，所述药物组合物的pH为5.5。在一些实施方案中，所述药物组合物的pH为约5.0。在一些实施方案中，所述药物组合物的pH为5.0。
在一些实施方案中，所述任一项所述的药物组合物的pH为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或6.0，或者为这些点值之间的任意范围。当本披露中提及点值时，应当理解该点值包含了误差范围。这种误差范围是由于实验室环境、人员操作、仪器、方法学、测量误差等因素所致。以pH为例，当测值为约5.0时，应当理解其包含了误差范围。作为一个示例，采用工业用pH计测量制剂时，“约5.0”表示5.0±0.2(即pH为4.8至5.2)。
在一些实施方式中，如前任一项所述的药物组合物，其中所述特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至250mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至150mg/mL。在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为5mg/mL至150mg/mL。在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至120mg/mL。在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至100mg/mL。在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为5mg/mL至100mg/mL。在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为70mg/mL至70mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为70mg/mL至150mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为80mg/mL至120mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为90mg/mL至110mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至20mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至10mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为4mg/mL至6mg/mL。
在一些实施方案中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为约1mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为约5mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为5mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为约70mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为70mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为约100mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为100mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为约150mg/mL。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为150mg/mL。
在一些实施方案中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL、4mg/mL、5mg/mL、6mg/mL、10mg/mL、20mg/mL、30mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、65mg/mL、70mg/mL、75mg/mL、80mg/mL、85mg/mL、90mg/mL、95mg/mL、100mg/mL、105mg/mL、110mg/mL、120mg/mL、130mg/mL、140mg/mL、150mg/mL、160mg/mL、170mg/mL、180mg/mL、190mg/mL、200mg/mL、210mg/mL、220mg/mL、230mg/mL、240mg/mL或250mg/mL，或者为这些点值之间的任意范围。在一些实施方案中，所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体的浓度为约1mg/mL、约4mg/mL、约5mg/mL、约6mg/mL、约10mg/mL、约20mg/mL、约30mg/mL、约40mg/mL、约45mg/mL、约50mg/mL、约55mg/mL、约60mg/mL、约65mg/mL、约70mg/mL、约75mg/mL、约80mg/mL、约85mg/mL、约90mg/mL、约95mg/mL、约100mg/mL、约105mg/mL、约110mg/mL、约120mg/mL、约130mg/mL、约140mg/mL、约150mg/mL、约160mg/mL、约170mg/mL、约180mg/mL、约190mg/mL、约200mg/mL、约210mg/mL、约220mg/mL、约230mg/mL、约240mg/mL或约250mg/mL。
在一些实施方案中，如前一项所述的药物组合物，其中所述药物组合物包含表面活性剂。在一些实施方案中，所述表面活性剂是非离子表面活性剂。在一些实施方案中，所述表面活性剂选自泊洛沙姆(例如P188)、聚山梨酯(例如聚山梨酯20、聚山梨酯80)、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等。
在一些实施方式中，如前所述表面活性剂为聚山梨酯或泊洛沙姆。在一些实施方式中，所述表面活性剂为聚山梨酯。在一些实施方式中，所述表面活性剂为聚山梨酯80或泊洛沙姆188(P188)。在一些实施方式中，所述表面活性剂为聚山梨酯80。
在一些实施方式中，如前任一项所述的药物组合物，其中所述聚山梨酯80的浓度为0.01mg/mL至1.0mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为0.1mg/mL至1.0mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为0.2mg/mL至0.6mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为0.3mg/mL至0.5mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为约0.1mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为0.1mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为约0.4mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为0.4mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为约1.0mg/mL。在一些实施方案中，所述聚山梨酯80的浓度为1.0mg/mL。在一些实施方案中，所述聚山梨酯80浓度为0.01mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL或1.0mg/mL，或者为这些点值之间的任意范围。在一些实施方案中，所述聚山梨酯80浓度为约0.01mg/mL、约0.05mg/mL、约0.1mg/mL、约0.15mg/mL、约0.2mg/mL、约0.3mg/mL、约0.4mg/mL、约0.5mg/mL、约0.6mg/mL、约0.7mg/mL、约0.8mg/mL、约0.9mg/mL或约1.0mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中所述泊洛沙姆188的浓度为0.5mg/mL至5mg/mL。在一些实施方案中，所述泊洛沙姆188的浓度为1mg/mL至3mg/mL。在一些实施方案中，所述泊洛沙姆188的浓度为1.5mg/mL至2.5mg/mL。在一些实施方案中，所述泊洛沙姆188的浓度为1.8mg/mL至2.2mg/mL。在一些实施方案中，所述泊洛沙姆188的浓度为约2mg/mL。在一些实施方案中，所述泊洛沙姆188的浓度为2mg/mL。在一些实施方案中，所述泊洛沙姆188浓度为0.5mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL、2mg/mL、2.1mg/mL、2.2mg/mL、2.3mg/mL、2.4mg/mL、2.5mg/mL、2.6mg/mL、2.8mg/mL、3mg/mL、3.5mg/mL、4mg/mL、4.5mg/mL或5.0mg/mL，或者为这些点值之间的任意范围。在一些实施方案中，所述泊洛沙姆188浓度为约0.5mg/mL、约0.8mg/mL、约0.9mg/mL、约1mg/mL、约1.5mg/mL、约1.6mg/mL、约1.7mg/mL、约1.8mg/mL、约1.9mg/mL、约2mg/mL、约2.1mg/mL、约2.2mg/mL、约2.3mg/mL、约2.4mg/mL、约2.5mg/mL、约2.6mg/mL、约2.8mg/mL、约3mg/mL、约3.5mg/mL、约4mg/mL、约4.5mg/mL或约5.0mg/mL。
在一些实施方案中，如前任一项所述的药物组合物，其包含糖。在一些实施方案中，所述糖选自常规组合物(CH2O)n及其衍生物，包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等。所述的糖可选自蔗糖，海藻糖，葡萄糖，乳糖，果糖，麦芽糖，右旋糖苷，甘油，赤藻糖醇，丙三醇，阿拉伯糖醇，sylitol，山梨糖醇，甘露醇，密二糖，松三糖，蜜三糖，甘露三糖，水苏糖，麦芽糖，乳果糖，麦芽酮糖，山梨醇，麦芽糖醇，乳糖醇，异-麦芽酮糖等。
在一些实施方式中，如前所述的糖为蔗糖、海藻糖、甘露醇或山梨糖醇。在一些实施方式中，所述糖为蔗糖。
在一些实施方式中，如前任一项所述的药物组合物，其中所述糖的浓度为10mg/mL至120mg/mL。在一些实施方式中，所述糖的浓度为30mg/mL至100mg/mL。在一些实施方式中，所述所述糖的浓度为64mg/mL至96mg/mL。在一些实施方案中，所述糖的浓度为68mg/mL至92mg/mL。在一些实施方案中，所述糖的浓度为72mg/mL至88mg/mL。在一些实施方案中，所述糖的浓度为约30mg/mL。在一些实施方案中，所述糖的浓度为约80mg/mL。在一些实施方案中，所述糖的浓度为约100mg/mL。在一些实施方案中，所述糖的浓度非限制性实施包括10mg/mL、20mg/mL、30mg/mL、35mg/mL、37.5mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、64mg/mL、65mg/mL、68mg/mL、70mg/mL、72mg/mL、75mg/mL、80mg/mL、85mg/mL、88mg/mL、90mg/mL、92mg/mL、95mg/mL、96mg/mL、100mg/mL或120mg/mL，以及这些点值之间的任一范围。在一些实施方案中，所述糖的浓度为30mg/mL。在一些实施方案中，所述糖的浓度为80mg/mL。在一些实施方案中，所述糖的浓度为100mg/mL。在一些实施方案中，所述糖的浓度非限制性实施包括约10mg/mL、约20mg/mL、约30mg/mL、约35mg/mL、约37.5mg/mL、约40mg/mL、约45mg/mL、约50mg/mL、约55mg/mL、约60mg/mL、约64mg/mL、约65mg/mL、约68mg/mL、约70mg/mL、约72mg/mL、约75mg/mL、约80mg/mL、约85mg/mL、约88mg/mL、约90mg/mL、约92mg/mL、约95mg/mL、约96mg/mL、约100mg/mL或约120mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中所述蔗糖的浓度为10mg/mL至120mg/mL。在一些实施方式中，所述蔗糖的浓度为30mg/mL至100mg/mL。在一些实施方式中，所述所述蔗糖的浓度为64mg/mL至96mg/mL。在一些实施方案中，所述蔗糖的浓度为68mg/mL至92mg/mL。在一些实施方案中，所述蔗糖的浓度为72mg/mL至88mg/mL。在一些实施方案中，所述蔗糖的浓度为约30mg/mL。在一些实施方案中，所述蔗糖的浓度为约80mg/mL。在一些实施方案中，所述蔗糖的浓度为约100mg/mL。在一些实施方案中，所述蔗糖的浓度非限制性实施包括10mg/mL、20mg/mL、30mg/mL、35mg/mL、37.5mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、64mg/mL、65mg/mL、68mg/mL、70mg/mL、72mg/mL、75mg/mL、80mg/mL、85mg/mL、88mg/mL、90mg/mL、92mg/mL、95mg/mL、96mg/mL、100mg/mL或120mg/mL，以及这些点值之间的任一范围。在一些实施方案中，所述蔗糖的浓度为30mg/mL。在一些实施方案中，所述蔗糖的浓度为80mg/mL。在一些实施方案中，所述蔗糖的浓度为100mg/mL。在一些实施方案中，所述蔗糖的浓度非限制性实施包括约10mg/mL、约20mg/mL、约30mg/mL、约35mg/mL、约37.5mg/mL、约40mg/mL、约45mg/mL、约50mg/mL、约55mg/mL、约60mg/mL、约64mg/mL、约65mg/mL、约68mg/mL、约70mg/mL、约72mg/mL、约75mg/mL、约80mg/mL、约85mg/mL、约88mg/mL、约90mg/mL、约92mg/mL、约95mg/mL、约96mg/mL、约100mg/mL或约120mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中所述缓冲剂的浓度为5mM至100mM。在一些实施方案中，所述缓冲剂的浓度为10mM至50mM。在一些实施方案中，所述缓冲剂的浓度为10mM至30mM。在一些实施方案中，所述缓冲剂的浓度为15mM至25mM。在一些实施方案中，所述缓冲剂的浓度为24mM至36mM。在一些实施方案中，所述缓冲剂的浓度为27mM至33mM。在一些实施方案中，所述缓冲剂的浓度为14mM至22mM。在一些实施方案中，所述缓冲剂的浓度为16mM至20mM。在一些实施方案中，所述缓冲剂的浓度为约30mM。在一些实施方案中，所述缓冲剂的浓度为约18mM。在一些实施方案中，所述缓冲剂的浓度为5mM、6mM、7mM、8mM、9mM、10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM、20mM、21mM、22mM、23mM、24mM、25mM、27mM、30mM、33mM、36mM、40mM、50mM、60mM、70mM、80mM、90mM或100mM，以及这些点值之间的任一范围。在一些实施方案中，所述缓冲剂的浓度为30mM。在一些实施方案中，所述缓冲剂的浓度为18mM。在一些实施方案中，所述缓冲剂的浓度为约5mM、约6mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、约15mM、约16mM、约17mM、约18mM、约19mM、约20mM、约21mM、约22mM、约23mM、约24mM、约25mM、约27mM、约30mM、约33mM、约36mM、约40mM、约50mM、约60mM、约70mM、约80mM、约90mM或约100mM。
在一些实施方式中，如前任一项所述的药物组合物，其中所述组氨酸-盐酸组氨酸缓冲剂的浓度为5mM至100mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为10mM至50mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为10mM至30mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为15mM至25mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为24mM至36mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为27mM至33mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为14mM至22mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为16mM至20mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为约30mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为约18mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为5mM、6mM、7mM、8mM、9mM、10mM、11mM、12mM、13mM、14mM、15mM、16mM、17mM、18mM、19mM、20mM、21mM、22mM、23mM、24mM、25mM、27mM、30mM、33mM、36mM、40mM、50mM、60mM、70mM、80mM、90mM或100mM，以及这些点值之间的任一范围。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为30mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为18mM。在一些实施方案中，所述组氨酸-盐酸组氨酸缓冲剂的浓度为约5mM、约6mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、约15mM、约16mM、约17mM、约18mM、约19mM、约20mM、约21mM、约22mM、约23mM、约24mM、约25mM、约27mM、约30mM、约33mM、约36mM、约40mM、约50mM、约60mM、约70mM、约80mM、约90mM或约100mM。
在一些实施方式中，如前任一项所述的药物组合物，其中所述药物组合物还包含辅料；优选地，所述辅料为二水合乙二胺四乙酸二钠、DTPA(二乙烯三胺五乙酸)、盐酸精氨酸、甘氨酸、甲硫氨酸、脯氨酸、组氨酸、苯丙氨酸、谷氨酸、天冬氨酸、氯化钠或氯化钙；更优选地，所述辅料为二水合乙二胺四乙酸二钠。本披露中乙二胺四乙酸二钠或二水合乙二胺四乙酸二钠含二水，简称EDTA-2Na，CAS号为：6381-92-6。
在一些实施方式中，如前任一项所述的药物组合物，其中所述辅料的浓度为0.01mg/mL至1mg/mL；优选地，辅料的浓度为0.01mg/mL至0.5mg/mL；更优选地，辅料的浓度为0.08mg/mL至0.12mg/mL。在一些实施方案中，所述辅料的浓度为0.01mg/mL至0.2mg/mL。在一些实施方案中，所述辅料的浓度为0.01mg/mL至0.12mg/mL。在一些实施方案中，所述辅料的浓度为0.01mg/mL至0.1mg/mL。在一些实施方案中，所述辅料的浓度为0.09mg/mL至0.11mg/mL。在一些实施方案中，所述辅料的浓度为约0.01mg/mL。在一些实施方案中，所述辅料的浓度为约0.1mg/mL。在一些实施方案中，所述辅料的浓度为约1mg/mL。在一些实施方案中，所述辅料的浓度为0.01mg/mL、0.03mg/mL、0.05mg/mL、0.08mg/mL、0.09mg/mL、0.1mg/mL、0.11mg/mL、0.12mg/mL、0.13mg/mL、0.14mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL或1mg/mL，以及这些点值之间的任一范围。在一些实施方案中，如前任一项所述的药物组合物，其中所述辅料的浓度为0.01mg/mL。在一些实施方案中，如前任一项所述的药物组合物，其中所述辅料的浓度为0.1mg/mL。在一些实施方案中，如请任一项所述的药物组合物，其中所述辅料的浓度为1mg/mL。在一些实施方案中，所述辅料的浓度为约0.01mg/mL、约0.03mg/mL、约0.05mg/mL、约0.08mg/mL、约0.09mg/mL、约0.1mg/mL、约0.11mg/mL、约0.12mg/mL、约0.13mg/mL、约0.14mg/mL、约0.15mg/mL、约0.2mg/mL、约0.3mg/mL、约0.4mg/mL、约0.5mg/mL、约0.6mg/mL、约0.7mg/mL、约0.8mg/mL、约0.9mg/mL或约1mg/mL。
在一些实施方案中，如前任一项所述的药物组合物，其中所述辅料为0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为0.01mg/mL至0.5mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为0.01mg/mL至0.2mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为0.01mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为0.01mg/mL至0.1mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为0.08mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为0.09mg/mL至0.11mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为约0.01mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为约0.1mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为约1mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述乙二胺四乙酸二钠的浓度为0.01mg/mL、0.03mg/mL、0.05mg/mL、0.08mg/mL、0.09mg/mL、0.1mg/mL、0.11mg/mL、0.12mg/mL、0.13mg/mL、0.14mg/mL、0.15mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL或1mg/mL，以及这些点值之间的任一范围。在一些实施方案中，所述辅料为0.01mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为0.1mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述辅料为1mg/mL的二水合乙二胺四乙酸二钠。在一些实施方案中，所述乙二胺四乙酸二钠的浓度为约0.01mg/mL、约0.03mg/mL、约0.05mg/mL、约0.08mg/mL、约0.09mg/mL、约0.1mg/mL、约0.11mg/mL、约0.12mg/mL、约0.13mg/mL、约0.14mg/mL、约0.15mg/mL、约0.2mg/mL、约0.3mg/mL、约0.4mg/mL、约0.5mg/mL、约0.6mg/mL、约0.7mg/mL、约0.8mg/mL、约0.9mg/mL或约1mg/mL。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中：
所述DLL3-VH包含如SEQ ID NO：33所示的氨基酸序列，和所述DLL3-VL包含如SEQ ID NO：42所示的氨基酸序列；和
所述CD3-VH包含SEQ ID NO：62的氨基酸序列，和所述CD3-VL包含SEQ ID NO：63的氨基酸序列。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中：
所述DLL3-VH的氨基酸序列如SEQ ID NO：33所示，和所述DLL3-VL的氨基酸序列如SEQ ID NO：42所示；和
所述CD3-VH的氨基酸序列如SEQ ID NO：62所示，和所述CD3-VL的氨基酸序列如SEQ ID NO：63所示。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体，其中所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中所述Fc1的氨基酸序列如SEQ ID NO：64所示；和所述Fc2的氨基酸序列如SEQ ID NO：65所示。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中所述Fc1的氨基酸序列如SEQ ID NO：66所示；和所述Fc2的氨基酸序列如SEQ ID NO：67所示。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中，所述的连接子1、连接子2和连接子3均为本领域已知的肽连接子，只要双特异性抗体能够展现出期望的抗原结合活性。例如，肽连接子可以是包含1-50或3-20个氨基酸残基的柔性肽。在一些实施方案中，所述肽连接子的长度为1-15个氨基酸残基。在一些实施方案中，所述连接子1具有如序列通式(GGGS)nGm所示的结构，其中n是1-5，优选为1、2或3；m为1-10，优选为4、5、6、7或8。在一些实施方案中，所述连接子1的序列为GGGSGGGG(SEQ ID NO：68)。在一些实施方案中，所述连接子2具有序列通式Gm所示的结构，其中m为1-10，优选为1-5，更优选为1、2或3。在一些实施方案中，连接子2的序列为G(SEQ ID NO：69)。在一些实施方案中，所述连接子3具有如序列通式(GGGGS)nGm所示的结构，其中n是1-5，优选为1、2或3；m为1-10，优选为4、5、6、7或8。在一些实施方案中，连接子3的序列为GGGGSGGGG(SEQ ID NO：70)。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中所述的连接子1、连接子2和连接子3相同或不同，氨基酸序列各自独立地选自SEQ ID NO：68、SEQ ID NO：69和SEQ ID NO：70。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中所述的连接子1、连接子2和连接子3的氨基酸序列依次为SEQ ID NO：68、SEQ ID NO：69和SEQ ID NO：70。
在一些实施方式中，如前任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中：
式I即链1的氨基酸序列如SEQ ID NO：71所示，和式II即链2的氨基酸序列如SEQ ID NO：72所示。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至250mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.01mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
(c)10mg/mL至120mg/mL的糖，和
(d)5mM至100mM的缓冲剂，所述药物组合物的pH为4.5至6.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至250mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.01mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
(c)10mg/mL至120mg/mL的糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)5mM至100mM的缓冲剂，所述药物组合物的pH为4.5至6.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
(c)30mg/mL至100mg/mL的蔗糖，和
(d)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂、组氨酸-醋酸组氨酸缓冲剂或枸橼酸-枸橼酸钠盐缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
(c)30mg/mL至100mg/mL的蔗糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂、组氨酸-醋酸组氨酸缓冲剂或枸橼酸-枸橼酸钠盐缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80或1mg/mL至3mg/mL泊洛沙姆188，
(c)30mg/mL至100mg/mL的蔗糖，和
(d)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80或1mg/mL至3mg/mL泊洛沙姆188，
(c)30mg/mL至100mg/mL的蔗糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)70mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80或1mg/mL至3mg/mL泊洛沙姆188，
(c)64mg/mL至96mg/mL的蔗糖，和
(d)10mM至30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)70mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80或1mg/mL至3mg/mL泊洛沙姆188，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.01mg/mL至0.5mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)80mg/mL至120mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.3mg/mL至0.5mg/mL的聚山梨酯80或1.5mg/mL至2.5mg/mL泊洛沙姆188，
(c)64mg/mL至96mg/mL的蔗糖，和
(d)15mM至25mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)80mg/mL至120mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.3mg/mL至0.5mg/mL的聚山梨酯80或1.5mg/mL至2.5mg/mL泊洛沙姆188，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.08mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠，和
(e)15mM至25mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至250mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.01mg/mL至1.0mg/mL的聚山梨酯80，
(c)10mg/mL至120mg/mL的糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)5mM至100mM的缓冲剂，所述药物组合物的pH为4.5至6.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80，
(c)30mg/mL至100mg/mL的蔗糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80，
(c)30mg/mL至100mg/mL的蔗糖，和
(e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)70mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.01mg/mL至0.5mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)80mg/mL至120mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.3mg/mL至0.5mg/mL的聚山梨酯80，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.08mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠，和
(e)15mM至25mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至250mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.5mg/mL至5mg/mL泊洛沙姆188，
(c)10mg/mL至120mg/mL的糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)5mM至100mM的缓冲剂，所述药物组合物的pH为4.5至6.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)1mg/mL至3mg/mL泊洛沙姆188，
(c)30mg/mL至100mg/mL的蔗糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)70mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)1mg/mL至3mg/mL泊洛沙姆188，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.01mg/mL至0.5mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)80mg/mL至120mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)1.5mg/mL至2.5mg/mL泊洛沙姆188，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.08mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠，和
(e)15mM至25mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，
(d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)约30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，
(d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)约30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为约5.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)0.4mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，
(d)0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，
(d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)约18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)0.4mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，
(d)0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，
(d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)约18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为约5.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)0.4mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，
(d)0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，和
(d)约30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)0.4mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，和
(d)30mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，和
(d)约18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)0.4mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，和
(d)18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，和
(d)约18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为约5.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)0.4mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，和
(d)18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约2mg/mL的泊洛沙姆188，
(c)约80mg/mL的蔗糖，
(d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)约18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)100mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)2mg/mL的泊洛沙姆188，
(c)80mg/mL的蔗糖，
(d)0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约70mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)约2mg/mL的泊洛沙姆188，
(c)约80mg/mL的蔗糖，
(d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)约18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为约5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)70mg/mL的特异性结合DLL3和CD3的双特异性抗体，
(b)2mg/mL的泊洛沙姆188，
(c)80mg/mL的蔗糖，
(d)0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至20mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.01mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
(c)10mg/mL至120mg/mL的糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)5mM至100mM的缓冲剂，所述药物组合物的pH为4.5至6.0；
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至10mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
(c)30mg/mL至100mg/mL的蔗糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂、组氨酸-醋酸组氨酸缓冲剂或枸橼酸-枸橼酸钠盐缓冲剂，所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至10mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.1mg/mL至1.0mg/mL的聚山梨酯80，
(c)30mg/mL至100mg/mL的蔗糖，
(d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)4mg/mL至6mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80或1mg/mL至3mg/mL泊洛沙姆188，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.01mg/mL至0.5mg/mL的二水合乙二胺四乙酸二钠，和
(e)10mM至30mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为4.5至5.5。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)4mg/mL至6mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.3mg/mL至0.5mg/mL的聚山梨酯80，
(c)64mg/mL至96mg/mL的蔗糖，
(d)0.08mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠，和
(e)15mM至25mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为4.8至5.2。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)约5mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)约0.4mg/mL的聚山梨酯80，
(c)约80mg/mL的蔗糖，
(d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)约18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为约5.0。
在一些实施方式中，如前任一项所述的药物组合物，其包含如下组分：
(a)5mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
(b)0.4mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，
(d)0.1mg/mL的二水合乙二胺四乙酸二钠，和
(e)18mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.0。
本领域技术人员应当理解，当参考数值范围、截止值或特定值使用时，“约”可以表示在1个或多于1个标准偏差之内。或者，“约”可以表示相差最高达20％的范围(即±20％)。由于本文所用的许多数值是通过实验确定的，因此本领域的技术人员应当理解，此类确定可以在不同实验之间有差异并且通常在不同实验之间有差异。由于这种内在差异，认为本文所用的值不应受到过度限制。因此，术语“约”用于涵盖与指定值相差±20％或更小的变化、±10％或更小的变化、±5％或更小的变化、±1％或更小的变化、±0.5％或更小的变化、或者±0.1％或更小的变化。
在一个方面，本披露提供一种制备冻干制剂的方法，其中包括将如前任一项所述的药物组合物进行冷冻干燥的步骤。
在一个方面，本披露提供一种冻干制剂，所述制剂通过如前所述的方法获得的。
在一个方面，本披露提供的如前任一项所述的药物组合物，其为皮下注射制剂、静脉注射制剂、腹腔注射制剂或肌肉注射制剂。在一些实施方案中，如上任一项所述的药物组合物、冻干制剂或复溶溶液，其为皮下注射制剂。
在一些实施方案中，如前任一项所述的药物组合物、冻干制剂或复溶溶液，其适用于皮下注射、静脉注射、腹腔注射或肌肉注射。在一些实施方案中，如上任一项所述的药物组合物、冻干制剂或复溶溶液，其适用于皮下注射。
在一些实施方案中，如前任一项所述的药物组合物、冻干制剂或复溶溶液，其用于制备皮下注射、静脉注射、腹腔注射或肌肉注射的药物。在一些实施方案中，如上任一项所述的药物组合物、冻干制剂或复溶溶液，其用于制备皮下注射的药物。
本披露还提供用作药物的如前任一项所述的药物组合物，或如前任一项所述的冻干制剂，或如前任一项所述的注射制剂。在一些实施方案中，所述药物用于治疗肿瘤或癌症。
本披露还提供一种治疗肿瘤或癌症的方法，所述方法包括向受试者施用治疗有效量的如前任一项所述的药物组合物，或如前任一项所述的冻干制剂，或如前任一项所述的注射制剂。
在一个方面，本披露提供如前任一项所述的组合物，或如前任一项所述的冻干制剂，或前任一项所述的注射制剂在制备用于治疗肿瘤或癌症的药物中的用途。
在一些实施方式中，如前任一项所述的肿瘤或癌症选自：肺癌、小细胞肺癌、大细胞肺癌、头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、咽鳞癌、口腔鳞癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、大细胞肺癌、三阴性乳腺癌和淋巴瘤。
在一些实施方式中，如前任一项所述的肿瘤或癌症是实体瘤。
在一些实施方式中，如前任一项所述的肿瘤或癌症是肺癌。
在一些实施方式中，如前任一项所述的肿瘤或癌症是小细胞肺癌。
在一些实施方案中，如前任一项所述的治疗肿瘤或癌症的方法，其进一步包括使用第二治疗剂。在一些实施方案中，所述第二治疗剂包括抗肿瘤药剂、放射疗法、抗体药物缀合物、双特异性抗体、与抗肿瘤药剂缀合的双特异性抗体、免疫检查点抑制剂或其组合。
在一些实施方案中，如前所述的治疗肿瘤或癌症的方法，其中所述的第二治疗剂与本披露任一项所述的双特异性抗体制剂同时施用、序贯施用或分开施用。
本披露提供的双特异性抗体制剂，具有治疗活性、安全性、药物代谢动力学特性和成药性(如稳定性)好的特点。
附图说明
图1显示DLL3-CD3双特异性抗体的Format3的结构示意图。
图2A显示双特异性抗体与DLL1结合的实验结果。结果显示，本披露的DLL3-CD3双特异性抗体不结合DLL1。
图2B显示双特异性抗体与DLL4结合的实验结果。结果显示，本披露的DLL3-CD3双特异性抗体不结合DLL4。
图3显示双特异性抗体对细胞的杀伤作用实验结果。结果显示，本披露的双特异性抗体对不表达DLL3的H460细胞无杀伤作用。
图4A显示双特异性抗体对T细胞的激活的实验结果，结果显示本披露的双特异性抗体在表达DLL3的DLL3/H82细胞存在时，具有明显的激活T细胞的作用。
图4B显示，当存在不表达DLL3的阴性细胞H460时，本披露的双特异性抗体不会激活T细胞。
图5A显示双特异性抗体的细胞因子释放的实验结果，结果显示，在H1184细胞存在的情况下，本披露的双特异性抗体刺激PBMC细胞分泌的IFNγ的含量非常低。
图5B显示，在H1184细胞存在的情况下，本披露的双特异性抗体刺激PBMC细胞分泌的IL-6的含量非常低。
具体实施方式
术语
本文所用的术语只是为了描述实施方案的目的，并非旨在进行限制。除非另外定义，本文所用的全部技术术语和科学术语具有与本披露所属领域的普通技术人员通常所理解的相同意义。
除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。除非另有说明，“包含”包括了“由……组成”。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“和/或”，例如“X和/或Y”应当理解为意指“X和Y”或“X或Y”并且应当被用来提供对两种含义或任一含义的明确支持。
除非被指定为来自非人物种(例如，“小鼠DLL3”、“小鼠DLL3片段”、“猴DLL3”、“猴DLL3片段”等)，否则如本文使用的“DLL3”和“DLL3片段”是指众所周知的人DLL3蛋白或其片段。
除非被指定为来自非人物种(例如，“小鼠CD3”、“小鼠CD3片段”、“猴CD3”、“猴CD3片段”等)，否则如本文使用的“CD3”和“CD3片段”是指众所周知的人CD3蛋白或其片段。
术语“氨基酸”是指天然存在的和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构(即与氢、羧基、氨基和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构，但是以与天然存在的氨基酸类似的方式起作用的化学化合物。
术语“氨基酸突变”包括氨基酸取代，缺失，插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来实现最终构建体，只要最终构建体拥有期望的特性，例如降低或对Fc受体的结合。氨基酸序列缺失和插入包括在多肽链的氨基端和/或羧基端的缺失和插入。具体的氨基酸突变可以是氨基酸取代。在一个实施方式中，氨基酸突变是非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸取代包括由非天然存在的氨基酸或由20种天然氨基酸的衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变。遗传方法可以包括定点诱变、PCR，基因合成等。预计基因工程以外的改变氨基酸侧链基团的方法，如化学修饰也是可用的。本文中可使用各种名称来指示同一氨基酸突变。本文中，可采用位置+氨基酸残基的方式表示特定位点的氨基酸残基，例如366W，则表示在366位点上的氨基酸残基为W。T366W则表示第366位点上的氨基酸残基由原来的T突变为了W。
术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗体片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。根据上下文，技术人员能够确定“抗体”所指的具体含义。
“天然抗体”指天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150000道尔顿的异四聚糖蛋白，由二硫键结合的两条轻链和两条重链构成。从N端至C端，每条重链具有一个可变区(VH，又称作可变重域、重链可变区)，接着是三个恒定域(CH1、CH2和CH3)。类似地，从N端至C端，每条轻链具有一个可变区(VL，又称作可变轻域，或轻链可变域)，接着是一个恒定轻域(轻链恒定区、CL)。术语“全长抗体”、“完整抗体”和“全抗体”在本文可互换使用，指具有与天然抗体结构基本类似的结构或具有含有如本文所限定的Fc区的重链的抗体。
术语“双特异性抗体”指能够对两个不同抗原或同一抗原的至少两个不同抗原表位特异性结合的抗体(包括抗体或其抗原结合片段，如单链抗体)。现有技术已公开了各种结构的双特异性抗体，根据IgG分子的完整性可分为IgG样双特异性抗体和抗体片段型双特异性抗体；根据抗原结合区域的数量，可分为二价、三价、四价或更多价的双特异性抗体，根据结构左右是否对称，可分为对称结构双特异性抗体和不对称结构双特异性抗体。其中，基于抗体片段的双特异性抗体，例如缺乏Fc片段的Fab片段，其通过将2个或多个Fab片段结合在一个分子中形成双特异性抗体，其具有较低的免疫原性，且分子量小，具有较高的肿瘤组织渗透性，该类型的典型的抗体结构如F(ab)2、scFv-Fab、(scFv)2-Fab等双特异性抗体；IgG样双特异性抗体(例如具有Fc片段)，这类抗体相对分子量较大，Fc片段有助于抗体后期的纯化，并提高其溶解性、稳定性，Fc部分还可能会与受体FcRn结合，增加抗体血清半衰期，典型的双特异性抗体结构模型如KiH、CrossMAb、Triomab quadroma、FcΔAdp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2：1TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab)2-CrossMAb、IgG-(scFv)2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab)4-CrossMAb等双特异性抗体(参见Aran F.Labrijn等，Nature Reviews Drug Discovery volume 18，pages585–608(2019)；Chen S1等，J Immunol Res.2019Feb 11；2019：4516041)。
可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018Oct 16；9:2278)等；各种编号系统之间的对应关系是本领域技术人员熟知的。本披露的编号规则如下表1中所示。
表1.CDR编号系统之间的关系

除非另有说明，本披露实施方案中的可变区和CDR序列均适用“Kabat”编号规则。
术语“抗体片段”或“抗原结合片段”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分保留了完整抗体的抗原结合能力。抗体片段的实例包括但不限于Fv、Fab、Fab’、Fab’-SH、F(ab′)2、单域抗体、单链Fab(scFab)、双抗体、线性抗体、单链抗体分子(例如scFv)；以及由抗体片段形成的多特异性抗体。
术语“Fc区”或“片段可结晶区”用于定义抗体重链的C末端区域，包括天然Fc区和改造的Fc区。在一些实施方式中，Fc区包含了相同或不同的两个亚基。在一些实施方式中，人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端。用于本文所述抗体的合适天然序列Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。除非另有说明，Fc区的编号规则为EU索引。
术语“嵌合”抗体指抗体中的重和/或轻链的一部分自特定的来源或物种衍生，而重和/或轻链的剩余部分自不同来源或物种衍生的抗体。
术语“人源化”抗体是保留非人抗体的反应性同时在人中具有较低免疫原性的抗体。例如，可以通过保留非人CDR区并用其人对应物(即，恒定区以及可变区的框架区部分)替换抗体的其余部分来实现。
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，“结合亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1相互作用。分子X对其配体Y的亲和力通常可以由平衡解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些)测量。术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合(association)速率，而如本文所使用的术语“kdis”或“kd”意在是指特定抗体-抗原相互作用的解离速率。如本文所使用的，术语“KD”指平衡解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域已知的方法测定抗体的KD值，例如表面等离子体共振法、ELISA或溶液平衡滴定法(SET)。
术语“单克隆抗体”指基本上均质的抗体的群，即在该群中包含的抗体分子的氨基酸序列是相同的，除了可能少量存在的天然突变以外。相比之下，多克隆抗体制剂通常包含在其可变结构域具有不同氨基酸序列的多种不同抗体，其通常特异性针对不同表位。在一些实施方式中，本披露提供的抗体是单克隆抗体。
术语“抗原”是指能够由抗原结合分子(例如抗体)的选择性结合剂所结合的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合分子(例如抗体)相互作用的表位。
术语“表位”指能够与抗体或其抗原结合片段特异性结合的抗原上的区域(area或region)。表位可以由连续氨基酸(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过蛋白质性质的抗原的三级折叠)而使得非连续的氨基酸在空间上得以接近。构象表位和线性表位的差别在于：在变性溶剂的存在下，抗体对构象表位的结合丧失。表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如但不限于丙氨酸扫描，肽印迹(见Meth.Mol.Biol.248(2004)443-463)，肽切割分析，表位切除，表位提取，抗原的化学修饰(见Prot.Sci.9(2000)487-496)，和交叉阻断(见“Antibodies”，Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harb.,NY))。
术语“能够特异性结合”、“特异性结合”或“结合”是指相比其他抗原或表位，抗原结合分子能够以更高的亲和力结合至某个抗原或表位。通常，抗体以约1×10-7M或更小(例如约1×10-8M或更小)的平衡解离常数(KD)结合抗原或表位。在一些实施方式中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％或更低(例如1％)。可使用已知的方法来测量KD，例如通过FACS或表面等离子体共振测定法所测量的。然而，特异性结合至抗原或其表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp))或狨猴(Callithrix jacchus)(commonmarmoset，marmoset)的相应抗原具有交叉反应性。
术语“不结合”是指抗原结合分子不能够以上述特异性结合的方式结合至某个抗原或其表位。例如，当抗体以约1×10-6M或更大的平衡解离常数(KD)结合抗原或其表位。
术语“抗原结合模块”指特异性结合目标抗原的多肽分子。具体的抗原结合模块包括抗体的抗原结合域，例如包含重链可变区和轻链可变区。术语“特异性结合CD3的抗原结合模块”是指能够以足够的亲和力结合CD3或其表位的模块，使得含有该模块的分子可用作靶向CD3的诊断剂和/或治疗剂。例如，特异性结合CD3的抗原结合模块具有以下的平衡解离常数(KD)：<约10nM，其是通过表面等离子体共振测定法测量的。抗原结合模块包括如本文定义的抗体片段，例如Fab，经替换的Fab或scFv。
术语“连接子”指连接两个多肽片段的连接单元。在本文中，同一结构式中出现的连接子可以是相同或不同的。连接子可以是肽连接子，其包含一个或多个氨基酸，典型的包含约1-30个、2-24个或3-15个氨基酸。应用于本文的连接子可以是相同或不同的。当“-”出现在结构式中，其表示两侧的单元直接通过共价键连接。
“Tm”是溶解变性温度(内源荧光)。当蛋白质变性(加热或变性剂作用)时，三级结构打开，芳香族氨基酸微环境发生变化，导致发射荧光光谱改变。本披露中，Tm1是指荧光变化到最大值的一半时的温度。
“Tonset”是变性起始温度。意指蛋白质开始变性时的温度，即荧光值开始变化时的温度。
“Tagg”是聚集起始温度。通过静态光散射，在266nm和473nm两个波长下检测聚集体，监测到样品开始聚集时的温度。Tagg 266指的是266nm下监测到聚集起始温度。
应当理解，尽管本公开中没有提供具体的编码核苷酸序列，技术人员根据密码子规则和宿主密码子偏好性，能够在氨基酸序列的基础上确定对应的编码核苷酸序列。
术语“融合”或“连接”是指部件(例如抗原结合模块和Fc结构域)直接地或经由连接子共价连接。
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。术语“表达载体”或“表达构建体”是指适用于对宿主细胞进行转化且含有指导及/或控制(连同宿主细胞一起)与其可操作地连接的一个或多个异源编码区的表达的核酸序列的载体。表达构建体可以包括但不限于影响或控制转录、翻译且在存在内含子时影响与其可操作地连接的编码区的RNA剪接的序列。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
术语“受试者”或“个体”包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。除非明确指出，否则所述术语“患者”或“受试者”在本文中可互换地使用。如本文所使用的，术语“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些实施方案中，个体或受试者是人。
“施用”或“给予”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。
术语“样本”是指从受试者分离的类似流体、细胞、或组织的采集物，以及存在于受试者体内的流体、细胞或组织。示例性样本为生物流体，诸如血液、血清和浆膜液、血浆、淋巴液、尿液、唾液、囊液、泪液、排泄物、痰、分泌组织和器官的粘膜分泌物、阴道分泌物、腹水、胸膜、心包、腹膜、腹腔和其它体腔的流体、由支气管灌洗液收集的流体、滑液、与受试者或生物来源接触的液体溶液，例如细胞和器官培养基(包括细胞或器官条件培养基)、灌洗液等，组织活检样本、细针穿刺、手术切除的组织、器官培养物或细胞培养物。
“治疗(treatment或treat)”和“处理”(及其语法变型)指试图施加至所治疗个体的临床干预，并且可以为了预防目的、或者在临床病理学的过程期间进行实施。治疗的期望效果包括但不限于预防疾病的发生或复发，减轻症状，减轻/减少疾病的任何直接或间接病理后果，预防转移，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。
术语“复发(recurrence)”、“复发(relapse)”“复发(relapsed)”是指癌症或疾病在疾病消失的临床评估之后的恢复。远处癌转移或局部复发的诊断可视为复发。
“有效量”一般是足以降低症状的严重程度及/或频率、消除这些症状及/或潜在病因、预防症状及/或其潜在病因出现及/或改良或改善由疾病状态引起或与其相关的损伤的量。在一些实施例中，有效量是治疗有效量或预防有效量。
“治疗有效量”是足以治疗疾病状态或症状、尤其与该疾病状态相关的状态或症状，或者以其他方式预防、阻碍、延迟或逆转该疾病状态或以任何方式与该疾病相关的任何其他不理想症状的进展的量。
“预防有效量”是当给予受试者时将具有预定预防效应，例如预防或延迟该疾病状态的发作(或复发)，或者降低该疾病状态或相关症状的发作(或复发)可能性的量。完全治疗或预防效未必在给予一个剂量之后便发生，可能在给予一系列剂量之后发生。因而，治疗或预防有效量可以一次或多次给予的方式给予。“治疗有效量”和“预防有效量”可取决于多种因素变化：诸如个体的疾病状态、年龄、性别和体重，以及治疗剂或治疗剂组合在个体中引发期望的应答的能力。有效治疗剂或治疗剂组合的示例性指标包括例如受试者改善的健康状况。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸缓冲剂”是包含组氨酸的缓冲剂。组氨酸缓冲剂的实例包括组氨酸-盐酸组氨酸，组氨酸-醋酸组氨酸，组氨酸-磷酸组氨酸，组氨酸-硫酸组氨酸等缓冲剂，优选组氨酸-盐酸组氨酸缓冲剂。组氨酸-盐酸组氨酸缓冲剂可由组氨酸与盐酸配制而成，或者由组氨酸与盐酸组氨酸配制而成。
“枸橼酸盐缓冲剂”是包括枸橼酸根离子的缓冲剂。枸橼酸盐缓冲剂的实例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁等。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠盐、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠盐。示例性的，所述的琥珀酸-琥珀酸钠可由琥珀酸与氢氧化钠配制而成，或由琥珀酸与琥珀酸钠盐配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括柠檬酸-磷酸氢二钠、磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选的磷酸盐缓冲剂是柠檬酸-磷酸氢二钠。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、组氨酸-醋酸组氨酸、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂是醋酸-醋酸钠。
“泊洛沙姆(poloxamer)”是α-氢-ω-羟基聚(氧乙烯)a-聚(氧丙烯)b-聚(氧乙烯)a嵌段共聚物。由环氧丙烷和丙二醇反应，形成聚氧丙烯二醇，然后加入环氧乙烷形成嵌段共聚物。其中，a为氧乙烯的单元数，b为氧丙烯的单元数。泊洛沙姆的实施例包括但不限于泊洛沙姆188(P188或PF68)。具体地，泊洛沙姆188，在共聚物中氧乙烯单元(a)为75～85，氧丙烯单元(b)为25～30，氧乙烯(EO)含量79.9％～83.7％，平均分子量为7680～9510。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
本披露所述的药物组合物能够达到一种稳定的效果：其中的抗体在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少1个月、至少3个月、优选6个月、更优选1年，且甚至更优选地多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃的温度保存包括1个月、3个月或6个月在内的时段后表现出期望的特征。还包括40℃的温度保存包括4周、1个月、3个月或6个月在内的时段后表现出期望的特征。稳定性的典型的例子：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体发生聚集或降解。通过视觉分析，制剂是淡黄色近无色澄明液体或者无色澄明液体，或澄清至稍微乳白色。所述制剂的浓度、pH、重量、分子渗透压具有不超过±10％变化，优选不超过±5％的变化。所述制剂通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和CE-SDS等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体在药物制剂中“保留它的生物活性”。
取代、插入和删除变体
在某些实施方案中，提供了具有一处或多处氨基酸取代的抗原结合分子变体。可以在CDR和FR进行取代。保守取代在表2中在“优选的取代”的标题下显示。更实质的变化在表2中在“示例性取代”的标题下提供，并且如下文参照氨基酸侧链类别进一步描述的。可以将氨基酸取代引入感兴趣的抗体中，并且对产物筛选期望的活性，例如保留/改善的抗原结合，降低的免疫原性，或改善的ADCC或CDC。
表2.氨基酸的取代

依照常见的侧链特性，氨基酸可以如下分组:
(1)疏水性的：正亮氨酸，Met，Ala，Val，Leu，Ile；
(2)中性，亲水性的：Cys，Ser，Thr，Asn，Gln；
(3)酸性的：Asp，Glu；
(4)碱性的：His，Lys，Arg；
(5)影响链取向的残基：Gly，Pro；
(6)芳香族的：Trp，Tyr，Phe。
非保守取代会是指用一个类别的成员替换另一个类别的成员。
一类取代变体涉及取代亲本抗体(例如人源化或人抗体)的一个或多个CDR残基。一般地，经选择用于进一步研究的所得变体相对于亲本抗体会具有某些生物学特性(例如升高的亲和力，降低的免疫原性)的改变(例如改善)，和/或会基本上保留亲本抗体的某些生物学特性。一种示例性的取代变体是亲和力成熟的抗体，可以例如使用基于噬菌体展示的亲和力成熟技术(如本文所述的那些技术)，便利地产生所述抗体。简言之，将一个或多个CDR残基突变，并将变体抗体在噬菌体上展示，并对其筛选特定的生物学活性(例如结合亲和力)。可以对CDR做出改变(例如取代)，例如以改善抗体亲和力。可以对CDR“热点”，即在体细胞成熟过程期间以高频率经历突变的密码子所编码的残基，和/或接触抗原的残基做出此类改变，同时对所得的变体VH或VL测试结合亲和力。在亲和力成熟的一些实施方案中，通过多种方法(例如易错PCR、链改组、或寡核苷酸指导的诱变)的任一种，将多样性引入所选择用于成熟的可变基因中。然后，创建次级文库。然后，筛选文库以鉴定具有期望的亲和力的任何抗体变体。另一种引入多样性的方法涉及CDR定向的方法，其中将几个CDR残基(例如一次4-6个残基)随机化。可以例如使用丙氨酸扫描诱变或建模来特异性鉴定涉及抗原结合的CDR残基。
在某些实施方案中，取代、插入或缺失可以在一个或多个CDR内发生，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对CDR做出保守变化(例如保守取代，如本文中提供的)，其不实质性降低结合亲和力。在上文提供的变体VH和VL序列的某些实施方案中，每个CDR是未改变的，或者含有不超过1、2或3处氨基酸取代。
一种可用于鉴定抗体中可以作为诱变靶位的残基或区域的方法称作“丙氨酸扫描诱变”。在这种方法中，鉴定一个残基或残基组(例如带电荷的残基，诸如Arg、Asp、His、Lys和Glu)，并且替换为中性或带负电荷的氨基酸(例如，Ala或聚丙氨酸)，以确定该抗体与抗原的相互作用是否受影响。可以在对初始取代显示功能敏感性的氨基酸位置引入进一步的取代。此外，可通过研究抗原-抗体复合物的晶体结构来鉴定抗体与抗原间的接触点。这些接触残基及邻近残基可以作为取代候选物被打靶或消除。可以筛选变体以确定它们是否含有期望的特性。
氨基酸序列插入包括：在氨基和/或羧基端融合1个残基或长度为100或更多个残基的多肽；和单个或多个氨基酸残基的序列内插入。在末端插入的例子包括具有N端甲硫氨酰基残基的抗体。抗体分子的其它插入变体包括，在抗体的N或C端融合有酶(或延长抗体的血清半衰期的多肽)的融合物。
Fc区的改造
在一个方面，本披露的双特异性抗体的Fc区包含一个或多个氨基酸取代，所述一个或多个氨基酸取代减少其与Fc受体的结合，例如其与Fcγ受体的结合，并且降低或消除效应子功能。天然IgG Fc区，具体地是IgG1 Fc区或IgG4 Fc区，可能导致本披露的双特异性抗体靶向表达Fc受体的细胞，而不是表达抗原的细胞。本披露改造的Fc区表现出降低的对Fc受体的结合亲和力和/或降低的效应子功能。在一些实施方案中，改造的Fc区与天然Fc区相比，对Fc受体的结合亲和力下降50％、80％、90％或95％以上。在一些实施方案中，所述的Fc受体是Fcγ受体。在一些实施方案中，所述Fc受体是人Fcγ受体，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些实施方案中，改造的Fc区与天然Fc区相比，对补体，如C1q的结合亲和力也降低。在一些实施方案中，改造的Fc区与天然Fc区相比，对新生儿Fc受体(FcRn)的结合亲和力不降低。在一些实施例中，改造的Fc区具有降低的效应子功能，所述降低的效应子功能可以包括但不限于以下中的一个或多个：降低的补体依赖性细胞毒性(CDC)、降低的抗体依赖性细胞介导的细胞毒性(ADCC)、降低的抗体依赖性细胞吞噬(ADCP)、减少的细胞因子分泌、减少的免疫复合物介导的抗原呈递细胞的抗原摄取、减少的与NK细胞的结合、减少的与巨噬细胞的结合、减少的与单核细胞的结合、减少的与多形核细胞的结合、减少的直接信号传导诱导性细胞凋亡、降低的树突细胞成熟或减少的T细胞引发。对于IgG1 Fc区，在238、265、269、270、297、327和329等位置的氨基酸残基取代可降低的效应子功能。在一些实施方案中，所述Fc区是人IgG1 Fc区，并且在234和235位置的氨基酸残基为A，编号依据为EU索引。对于IgG4 Fc区，在228等位置的氨基酸残基取代可降低的效应子功能。
当双特异性抗体包含与Fc区的两个亚基融合的不同结合模块，可能导致不期望的同源二聚化。为了提高产率和纯度，因此在本披露的双特异性抗体的Fc区中引入促进异源二聚化的修饰将是有利的。在一些实施方式中，本披露的Fc区包含根据杵臼(knob-into-hole，KIH)技术的改造，该方法涉及在第一亚基的界面处引入凸起结构(knob)以及在第二亚基的界面处引入孔结构(hole)。使得所述凸起结构可以定位在孔结构中，促进异源二聚体的形成并抑制同源二聚体的产生。凸起结构是通过用较大侧链(例如酪氨酸或色氨酸)取代来自第一亚基的界面的小氨基酸侧链而构建的。而孔结构是通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大氨基酸侧链而在第二亚基的界面中创建的。凸起结构和孔结构通过改变编码多肽的核酸来制备，可选的氨基酸取代如下表所示：
表3.KIH突变组合
除了杵臼技术外，用于修饰重链的CH3结构域以实现异源二聚化的其他技术也是本领域中已知的，例如WO1996027011A1、WO1998050431、EP1870459、WO2007110205、WO2009089004、WO2010129304、WO201190754、WO2011143545、WO2012058768、WO2013157954和WO2013096291。
Fc区的C末端可以是以氨基酸残基PGK结束的完整C末端；也可以是截短的C末端，例如在所述截短的C末端中去除了一个或两个C末端氨基酸残基。在一个优选的方面中，重链的C末端是以PG结束的缩短的C末端。因此，在一些实施方式中，完整抗体的组合物可以包括去除了所有K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物可以包括没有去除K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物具有带有和不带有K447残基和/或G446+K447残基的抗体混合物的抗体群体。
重组方法
双特异性抗体可以使用重组方法来产生。对于这些方法，提供编码双特异性抗体的一个或更多个分离的核酸。
在天然抗体、天然抗体片段或具有同源二聚体重链的双特异性抗体的情况下，需要两个核酸，一个用于轻链或其片段，一个用于重链或其片段。此类核酸编码包含抗体VL的氨基酸序列和/或包含抗体VH的氨基酸序列(例如抗体的轻链和/或重链)。这些核酸可以在相同的表达载体上或在不同的表达载体上。
在具有异二聚体重链的双特异性抗体的情况下，需要例如四个核酸，一个用于第一轻链，一个用于包含第一异源单体Fc区多肽的第一重链，一个用于第二轻链，并且一个用于包含第二异源单体Fc区多肽的第二重链。这四个核酸可包含在一个或更多个核酸分子或表达载体中，通常这些核酸位于两个或三个表达载体上，即一个载体可包含这些核酸中的多于一个。
在一个实施方案中，本披露提供了编码如前所述的抗体的分离的核酸。此类核酸可以独立地编码前述的任一多肽链。在另一方面中，本披露提供了包含此类核酸的一种或多种载体(例如表达载体)。在另一方面中，本披露提供了包含此类核酸的宿主细胞。在一个实施方案中，提供制备双特异性抗体的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体的核酸的宿主细胞，如上文所提供的，和任选地从宿主细胞(或宿主细胞培养基)回收所述抗体。
为了重组产生双特异性抗体，将编码蛋白的核酸分离并插入一个或更多个载体中，用于在宿主细胞中进一步克隆和/或表达。此类核酸可以使用常规程序容易地分离和测序(例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针)，或者通过重组方法产生或通过化学合成获得。
用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，抗体可在细菌中产生，特别是当抗体不需要糖基化和Fc效应子功能时。在表达后，抗体可以在可溶级分中从细菌细胞糊状物分离，并且可进一步纯化。
除了原核生物以外，真核微生物诸如丝状真菌或酵母也是用于编码抗体的载体的合适的克隆或表达宿主，包括真菌和酵母菌株，其糖基化途径已经“人源化”，导致产生具有部分或完全人糖基化模式的抗体。适于表达(糖基化)抗体的合适的宿主细胞也可源自多细胞生物体(无脊椎动物和脊椎动物)；无脊椎动物细胞的例子包括植物和昆虫细胞。已经鉴定了许多杆状病毒株，其可与昆虫细胞联合使用，特别是用于草地贪夜蛾(Spodoptera frugiperda)细胞的转染；还可利用植物细胞培养物作为宿主，例如US5959177、US 6040498、US6420548、US 7125978和US6417429；也可将脊椎动物细胞用作宿主，例如适应于在悬浮液中生长的哺乳动物细胞系。适宜的哺乳动物宿主细胞系的其它例子是经SV40转化的猴肾CVl系(COS-7)；人胚肾系(293或293T细胞)；幼仓鼠肾细胞(BHK)；小鼠塞托利(sertoli)细胞(TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人宫颈癌细胞(HELA)；犬肾细胞(MDCK)；水牛鼠(buffalo rat)肝细胞(BRL3A)；人肺细胞(W138)；人肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞；MRC 5细胞；和FS4细胞。其它适宜的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞；以及骨髓瘤细胞系，如Y0、NS0和Sp2/0。关于适合产生抗体的某些哺乳动物宿主细胞系的综述参见例如Yazaki，P.和Wu，A.M.，Methods in Molecular Biology，Vol.248，Lo，B.K.C.(编)，Humana Press，Totowa，NJ(2004)，第255-268页。
实施例与测试例
以下结合实施例和测试例进一步描述本披露，但这些实施例和测试例并非限制着本披露的范围。本披露实施例和测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
一、特异性结合DLL3和CD3的双特异性抗体的制备
实施例1.DLL3抗原及检测用蛋白和稳转细胞株的制备
1.1高表达DLL3的细胞株构建
包含SEQ ID NO：1-2的pCDH慢病毒表达载体质粒(由GENEWIZ公司合成)，pCDH质粒分别与pVSVG，pCMV慢病毒包装载体用Lipofectamine 3000(Invitrogen,L3000015)转染试剂转染至293T细胞(中科院细胞库,GNHu17)中，收集含有病毒的培养基上清，过滤并进行超高速离心，弃去上清后，用0.2mL无菌PBS重悬。使用浓缩后的病毒分别感染中国仓鼠卵巢细胞CHO-s(Invitrogen,R80007)，DMS53(ATCC,CRL-2062)和H82(ATCC,HTB-175)，经嘌呤霉素(puromycin)筛选两至三周，再进行FACS单细胞分选。将挑选出的单克隆细胞株扩大培养，冻存，以便后续实验。
包含表达LUC和GFP基因的pCDH-CMV-MCS-EF1-puro慢病毒表达载体质粒(由GENEWIZ公司合成)，pCDH质粒分别与pVSVG，pCMV慢病毒包装载体用Lipofectamine 3000(Invitrogen,L3000015)转染试剂转染至293T细胞(中科院细胞库,GNHu17)中，收集含有病毒的培养基上清，过滤并进行超高速离心，弃去上清后，用0.2mL无菌PBS重悬。使用浓缩后的病毒分别感染SHP77(ATCC,CRL-2195)，H1184(ATCC，CRL-5858)，H460(中国科学院细胞库，TCHu205)经嘌呤霉素(puromycin)筛选两至三周，再进行FACS单细胞分选。将挑选出的单克隆细胞株扩大培养，冻存，以便后续实验。
使用的相关蛋白序列如下：
1.人DLL3全长蛋白(SEQ ID NO:1)：

2.食蟹猴DLL3全长蛋白(SEQ ID NO:2)：
3.大鼠DLL3全长蛋白(SEQ ID NO:3)：
4.小鼠DLL3全长蛋白(SEQ ID NO:4)：

5.人DLL1全长蛋白(SEQ ID NO:5)：
6.人DLL4全长蛋白(SEQ ID NO:6)：

1.2抗原的制备
以人DLL3(Uniprot,Q9NYJ7)、食蟹猴DLL3(Uniprot,A0A2K5WSR4)和小鼠DLL3(Uniprot,O88516)序列为模板，设计含有不同标签的人DLL3 ECD融合蛋白，分别克隆到pTT5载体上，经293E细胞表达后，获得抗原。相关蛋白氨基酸序列如下：
1.His-hDLL3(ECD)(SEQ ID NO:7)：
注：点划线为信号肽序列，单划线为his标签和连接子，双划线为DLL3胞外区。
2.Fc-hDLL3(ECD)(SEQ ID NO:8)：
注：点划线为信号肽序列，单划线为Fc标签和连接子，双划线为DLL3胞外区。
3.hDLL3(ECD)-strep twin(SEQ ID NO:9)：
注：点划线为信号肽序列，双划线为DLL3胞外区，单划线为strep twin标签。
4.cynoDLL3(ECD)-strep twin(SEQ ID NO:10)：
注：点划线为信号肽序列，双划线为DLL3胞外区，单划线为strep twin标签。
5.mouDLL3(ECD)-strep twin(SEQ ID NO:11)：
注：点划线为信号肽序列，双划线为DLL3胞外区，单划线为strep twin标签。
实施例2.抗人DLL3抗体的制备
通过杂交瘤技术制备针对人DLL3的单克隆抗体，免疫方式如下：
第一组，以His-hDLL3(ECD)(SEQ ID NO：7)为免疫原，用Gold Adjuvant(Sigma Cat No.T2684)与ThermoAlum(Thermo Cat No.77161)佐剂交叉免疫。抗原与佐剂(Gold Adjuvant)比例为1:1，抗原与佐剂(ThermoAlum)比例为3:1，50μg/只/次(首次免疫)，25μg/只/次(加强免疫)。用ELISA方法确定小鼠血清中的抗体滴度。
第二组小鼠的免疫抗原为DLL3-CHO-s细胞和Fc-hDLL3(ECD)(SEQ ID NO：8)，免疫方式为细胞和蛋白抗原交替免疫。细胞免疫按照每只小鼠腹膜内注射0.1mL生理盐水稀释至108/mL浓度的细胞液。Fc-hDLL3(ECD)抗原用Gold Adjuvant(Sigma Cat No.T2684)与ThermoAlum(Thermo Cat No.77161)佐剂交叉免疫。抗原与佐剂(Gold Adjuvant)比例为1:1，抗原与佐剂(ThermoAlum)比例为3:1，50μg/只/次(首次免疫)，25μg/只/次(加强免疫)。用ELISA方法确定小鼠血清中的抗体滴度。
采用优化的电融合方法将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(CRL-8287TM)进行融合得到杂交瘤细胞。根据杂交瘤细胞生长密度，用结合DLL3蛋白的ELISA方法和结合DLL3 CHO-s细胞FACS方法进行杂交瘤培养上清检测。选择结合人DLL3蛋白，猴DLL3蛋白以及DLL3 CHO-s细胞，同时不结合野生型CHO-s细胞的克隆及时进行冻存扩增保种和一到二次亚克隆直至获得单细胞克隆。通过以上实验筛选得到杂交瘤克隆mAb6。
将杂交瘤克隆扩培养，提取RNA，利用mouse-Ig的简并引物进行反转录扩增(RT-PCR)，最后得到抗体的可变区序列。
mAb6重链可变区(SEQ ID NO:12)：
mAb6轻链可变区(SEQ ID NO:13)：
表4.抗体的CDR

注：VH/VL的CDR的氨基酸残基由Kabat编号系统确定并注释。
将鼠抗体的重链可变区和轻链可变区分别克隆到包含SEQ ID NO:20所示的人IgG1重链恒定区和SEQ ID NO:21所示的κ轻链恒定区的pTT5载体质粒，然后转染HEK293细胞，得到了抗DLL3的嵌合抗体M6CHI。
人IgG1重链恒定区(SEQ ID NO：20)：
人κ轻链恒定区(hκ)(SEQ ID NO：21)：
实施例3.抗DLL3单克隆抗体的人源化
通过比对Kabat人类抗体重轻链可变区种系基因数据库，分别挑选同源性高的重、轻链可变区种系基因作为模板，将鼠源抗体的CDR分别嫁接(graft)到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。并对可变区的某些氨基酸进行氨基酸取代，再与恒定区重组(示例性的，与SEQ ID NO：20所示的人IgG1重链恒定区和SEQ ID NO：21所示的人κ轻链恒定区)，获得全长的人源化抗体。
mAb6抗体的人种系轻链模版为IGKV1-16*01或IGKV3-20*02和IGKJ4*01，人种系重链模版为IGHV1-3*01、IGHV7-4-1*02或IGHV3-73*01和IGHJ6*01。任选地，对人源化抗体的轻链可变区上第1、36、42、43、44、46、52、56、69、71、79、85和/或94位的氨基酸残基进行取代；和/或对人源化抗体的重链可变区上第1、27、30、38、43、48、67、68、69、71、73、75、76、93、56、58和/或5位的氨基酸残基进行取代。
表5.mAb6鼠源抗体的人源化模板及相应点突变


注：表中的氨基酸位置按照为kabat编号，F36L表示依照Kabat编号系统，将36位F突变为L下同。
获得的hu6人源化抗体可变区的序列如下：
hu6 VH1(SEQ ID NO:22)
hu6 VH2(SEQ ID NO:23)
hu6 VH3(SEQ ID NO:24)
hu6 VH4(SEQ ID NO:25)
hu6 VH5(SEQ ID NO:26)
hu6 VH6(SEQ ID NO:27)
hu6 VH7(SEQ ID NO:28)
hu6VH8(SEQ ID NO:29)(Graft IGHV1-3*01)
hu6VH9(SEQ ID NO:30)(Graft IGHV7-4-1*02)
hu6VH10(SEQ ID NO:31)(Graft IGHV3-73*01)
hu6VH11(SEQ ID NO:32)
hu6VH12(SEQ ID NO:33)
hu6VH13(SEQ ID NO:34)
hu6VH14(SEQ ID NO:35)
hu6VH15(SEQ ID NO:36)
hu6 VL1(SEQ ID NO:37)
hu6 VL2(SEQ ID NO:38)
hu6 VL3(SEQ ID NO:39)
hu6 VL4(SEQ ID NO:40)
hu6VL5(SEQ ID NO:41)(Graft IGKV1-16*01)
hu6VL6(SEQ ID NO:42)
hu6VL7(SEQ ID NO:43)
hu6VL8(SEQ ID NO:44)
hu6VL9(SEQ ID NO:45)
hu6VL10(SEQ ID NO:46)
Graft(IGKV3-20*02)+Y36L,Q42K,L46G,T69A,F71Y,E79Q,V85D+D56E
hu6VL11(SEQ ID NO:47)Graft(IGKV3-20*02)+D56E
注：单下划线部分为CDR，双下划线处为氨基酸取代位点，其余部分为FR区，下同。
mAb6的人源化抗体的CDR如下：
表6.mAb6人源化抗体的CDR
表7.mAb6的人源化抗体

注：hu6L1H1表示该抗体包含重链可变区hu6VH1和轻链可变区hu6VL1，且其重链恒定区的序列为SEQ ID NO：20，轻链恒定区的序列为SEQ ID NO：21，其他类推。
表8.mAb6的人源化抗体
分别克隆、表达、纯化上述抗体，经蛋白结合实验(测试例3)、细胞结合实验(测试例2)、Biacore(测试例1)，最终选出活性较好的人源化抗体。示例性的人源化抗体的重、轻链氨基酸序列如下：
Hu6(也称hu6L4H12)重链(SEQ ID NO：54)：

Hu6(也称hu6L4H12)轻链(SEQ ID NO：55)：
注：序列中下划线部分为可变区，斜体部分为恒定区。
实施例4.抗DLL3-CD3双特异性抗体的制备
4.1本披露使用的CD3抗体
本披露的CD3结合部分可以来源于任意适宜的抗体。具体的，本披露的实施例采用的是S107E，其可变区及CDR序列如下：
表9.S107E的CDR
S107E可变区序列如下：
>S107E-VH(SEQ ID NO：62)
>S107E-VL(SEQ ID NO：63)
注：下划线部分为CDR。
4.2本披露的DLL3-CD3双特异性抗体的结构
本披露的DLL3-CD3双特异性抗体分子包含两条链，具体结构如下：
链1结构：[VL(anti-DLL3)]-连接子1-[VH(anti-CD3)]-连接子2-[Fc(Knob,L234A，L235A，G237A，Y349C，T366W)]；
链2结构：[VL(anti-CD3)]-连接子3-[VH(anti-DLL3)]-连接子2-[Fc(hole,L234A，L235A，G237A，S354C，T366S，L368A，Y407V)]；
其示意图如图1所示。
双特异性抗体分子两条链的Fc区为能够以杵臼技术相互缔合的Fc区。示例性的，链1的Fc区为具有根据杵臼技术的凸起结构(如SEQ ID NO：64或SEQ ID NO：66)，链2的Fc区为具有根据杵臼技术的孔结构(如SEQ ID NO：65或SEQ ID NO：67)。
相关序列如下：
>IgG1Fc(Knob,L234A/L235A/S354C/T366W)(SEQ ID NO：64)
>IgG1Fc(Hole,L234A,L235A,Y349C,T366S,L368A,Y407V)(SEQ ID NO：65)
>Fc Knob(L234A，L235A，G237A，Y349C，T366W)(SEQ ID NO：66)
>Fc hole(L234A,L235A，G237A，S354C，T366S，L368A，Y407V)(SEQ ID NO：67)
>连接子1(SEQ ID NO：68)
>连接子2(SEQ ID NO：69)
>连接子3(SEQ ID NO：70)
基于mAb6人源化抗体氨基酸序列，构建了如下所示的双特异性抗体：hu6(D56E)-S107E-diabody
链1：hu6-VL(D56E)-连接子1-S107E-连接子2-VH-Fc knob；(SEQ ID NO：71)
链2：CD3VL-连接子3-hu6VH-连接子2-Fc hole；(SEQ ID NO：72)
注：hu6(D56E)-S107E-diabody表示该分子采用hu6(D56E)人源化抗体的可变区作为DLL3结合域，S107E的可变区作为CD3结合域，采用diabody作为分子结构。
>链1(SEQ ID NO：71)
>链2(SEQ ID NO：72)
本披露中使用的对照分子AMG-75，其参考WO2017021349制备。对照分子的序列如下：
AMG-757(SEQ ID NO：73):
测试例
测试例1：Biacore抗体亲和力测定
用Protein A生物传感芯片(Cat.#29127556，Cytiva)亲和捕获待测抗体18秒，然后于芯片表面流经抗原人DLL3(ACRO，DLL3-H52H4)，食蟹猴DLL3(KACTUS，DLL-RM103)，小鼠DLL3(KACTUS，DLL-MM103)，人CD3D&3E(ACRO，CDD-H52W1)和猴CD3D&3E(ACRO，CDD-C52W4)180秒，然后解离600秒。用Biacore 8K(Cytiva)仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用10mM甘氨酸-盐酸溶液(pH 1.5，Cat.#BR-1003-54，Cytiva)将芯片洗净再生。数据拟合模型采用1:1Model。结果见表10和表11。
表10.抗体与人DLL3的亲和力(基于克隆6)

表11.双特异性抗体hu6(D56E)-S107E-diabody与不同种属抗原的亲和力
结果显示，本披露的特异性结合DLL3的人源化抗体，嵌合抗体与人DLL3均具有良好亲和力；双特异性抗体与人和食蟹猴的DLL3和CD3均具有良好的亲和力。
测试例2：DLL3细胞水平的FACS结合实验
将表达DLL3的小细胞肺癌细胞系H1184(ATCC，货号CRL-5858)，DLL3/H82，DLL3/SHP77，hDLL3/CHO-s，cynoDLL3/CHO-s细胞，表达CD3的细胞Jurkat(ATCC，TIB-152)用FACS缓冲液(1％BSA+pH 7.4PBS)制备成1×106/mL的细胞悬液，100μL/孔加入96孔圆底板中。300g离心5分钟，去除上清。加入不同浓度待测抗体，100μL/孔。放于4℃冰箱中避光孵育1小时。300g离心洗涤3次后，加入工作浓度的APC anti-human IgG Fc(BioLegend，410712)或者PE F(ab')2-羊抗人IgG(invitrogen,H10104)，放于4℃冰箱中避光孵育40分钟。300g离心洗涤3次后，在Invitrogen流式细胞仪上检测几何平均数荧光强度，计算抗体对表达DLL3的细胞的结合EC50值。结果见表12和表13。
表12.抗体与表达人DLL3的H1184细胞的结合活性
表13.双特异性抗体与细胞的结合活性
结果显示，本披露的抗DLL3人源化抗体与表达人DLL3的细胞具有良好结合能力；双特异性抗体对表达人和食蟹猴的DLL3细胞以及CD3的细胞有良好的结合能力。
测试例3：DLL3蛋白水平的ELISA结合实验
将streptavidin(abcam，ab136200，1μg/ml)包板，100μL/孔，4℃过夜。用PBST溶液(PBS含0.1％吐温20)250μL/孔，洗板3次。5％牛奶封闭，250μL/孔，37℃封闭2小时。用PBST溶液250μL/孔，洗板3次。加入生物素化的DLL3抗原(1μg/mL)(SEQ ID NO：9)，37℃孵育1小时。使用PBST溶液250μL/孔，洗板3次。配制抗体(最高浓度400nM，4倍梯度稀释)，37℃孵育1小时。使用PBST溶液250μL/孔，洗板6次。加入工作浓度的human IgG(H+L)-HRP(Jackson,109-035-003，1：4000稀释)抗体，100μL/孔，37℃孵育1小时。使用PBST溶液250μL/孔，洗板6次。加入TMB(KPL,5120-0077)显色液100μL/孔，室温显色5-10分钟。加入1M H2SO4，100μL/孔，中止显色，酶标仪(Molecular Devices,VERSA max)450nm读数。
表14.双特异性抗体与人DLL3蛋白的结合活性
结果显示，本披露的双特异性抗体对人DLL3蛋白有良好的亲和力。
测试例4：DLL1和DLL4蛋白的ELISA结合实验
将DLL1(ACRO，DL1-H52H8，1μg/mL)和DLL4(ACRO，DL4-H5227，1μg/mL)包板，100μL/孔，4℃过夜。用PBST溶液(PBS含0.1％吐温20)250μL/孔，洗板3次。5％牛奶封闭，250μL/孔，37℃封闭2小时。用PBST溶液250μL/孔，洗板3次。加入待检测抗体(最高浓度100nM，4倍梯度稀释)，37℃孵育1小时。使用PBST溶液250μL/孔，洗板6次。加入工作浓度的human IgG(H+L)-HRP(Jackson,109-035-003，1：4000稀释)抗体，100μL/孔，37℃孵育1小时。使用PBST溶液250μL/孔，洗板6次。加入TMB(KPL，5120-0077)显色液100μL/孔，室温显色5-10分钟。加入1M H2SO4，100μL/孔，中止显色，酶标仪(Molecular Devices,VERSA max)450nm读数，结果见图2A-图2B。
结果显示，本披露的双特异性抗体未检测到结合人DLL1和DLL4蛋白。
测试例5.本披露的双特异性抗体对细胞的杀伤活性
本测试例研究了双特异性抗体作为T细胞衔接分子对肿瘤细胞的杀伤活性。用表达LUC-GFP的SHP77、H1184和H460稳转细胞系作为靶细胞来检测本披露的双特异性抗体的靶点特异性细胞毒活性。
SHP77/lucG细胞培养在1640+10％ FBS+10μg/mL嘌呤霉素(puromycin)完全培养基中，一周传代2-3次。H1184/lucG细胞培养在1640+5％ FBS+10μg/mL嘌呤霉素(puromycin)完全培养基中，一周传代1次。H460/lucG细胞培养在1640+10％FBS完全培养基中，一周传代2-3次。冻存PBMC(妙顺生物)复苏后用1640+10％FBS完全培养基重悬，置于T75培养瓶培养4小时(密度为2E6 cells/mL)。PBMC收集离心后用1640+10％ FBS重悬、计数，调整细胞数为1.5E6 cells/mL。将上述PBMC悬液和靶细胞悬液按照等体积混合，每孔加入100μL确保E:T Ratio为10:1。另外设置PBMC ONLY组，将上述PBMC悬液和1640+10％ FBS完全培养基按照等体积混合。抗体用1640+10％ FBS培养基稀释，起始浓度为600nM(6x终浓度)，5倍稀释，9个剂量点,每孔加入20μL。处理好的细胞放在37℃,5％ CO2的培养箱培养48小时。取出培养板，1000rpm离心3分钟，吸取50μL上清于新的3788板中用于检测细胞因子检测，-20℃保存。在培养板中加入50μLONE-GloTMLuciferase(Promega，E6120)，室温孵育5分钟后，用Vendor检测Luminescence，计算抗体在不同浓度下的杀伤作用。使用Graphpad Prism8.0软件根据抗体的对数浓度和信号值做出量效曲线图，得出抗体介导杀伤的IC50。将只有靶细胞、PBMC不加抗体的孔设为0％抑制，计算抗体的最大抑制率Imax。结果见表15及图3。
表15.双特异性抗体hu6(D56E)-S107E-diabody对不同靶细胞的细胞毒活性
结果显示，本披露的双特异性抗体对表达DLL3的细胞SHP77和H1184有较强的杀伤作用；而对不表达DLL3的H460细胞无杀伤作用(图3)。
测试例6.本披露的双特异性抗体的体外T细胞激活活性
H82/DLL3细胞培养在1640+10％ FBS完全培养基中，一周传代2-3次，传代比例1:3左右。H460细胞培养在1640+10％ FBS完全培养基中，一周传代2-3次，传代比例1:10左右。Jurkat LuciaTMNFAT细胞(InvivoGen)培养在1640+10％FBS+100μg/ml zeocin(InvivoGen,ant-zn-1)完全培养基中，一周传代2-3次，传代比例1:10左右。收集Jurkat LuciaTMNFAT细胞，1000rpm离心3分钟，重悬、计数，调整细胞数为1E6 cells/mL。收集靶细胞H82/DLL3细胞，1000rpm离心3分钟，重悬、计数，调整细胞数为2E5 cells/mL。收集H82/DLL3和H460细胞，1000rpm离心3分钟，重悬、计数，调整细胞数为4E5 cells/mL。将上述Jurkat LuciaTMNFAT和靶细胞按照等体积混合，每孔加入100μL，确保H82/DLL3细胞E:T Ratio为5:1，H82/DLL3和H460细胞E:T Ratio为2.5:1。抗体用1640+10％ FBS培养基稀释，起始浓度为600nM(6×终浓度)，5倍稀释，9个剂量点,每孔加入20μL。处理好的细胞放在37℃,5％ CO2的培养箱培养5小时。用25mL的ddH2O溶解QUANTI-LucTMGold试剂(Invivogen,rep-qlcg5)。加入50μL QUANTI-LucTMGold，室温孵育5分钟后，用Vendor检测Luminescence，计算抗体在不同浓度下对Jurkat LuciaTMNFAT细胞的激活作用。使用Graphpad Prism8.0软件做图，结果见图4A至图4B和表16。
激活倍数的公式N＝待测抗体的荧光读值/阴性孔的荧光读值
表16.双特异性抗体对T细胞的激活结果
结果显示，本披露的双特异性抗体只有在存在表达DLL3的DLL3/H82细胞时，才会激活T细胞；当存在不表达DLL3的阴性细胞H460时，不会激活T细胞。
测试例7.本披露的双特异性抗体的细胞因子释放水平
1.本披露的双特异性抗体的细胞因子IFNγ释放水平
使用HTRF法检测在DLL3/CD3双抗在H1184细胞存在的情况下，分别刺激PBMC中细胞因子IFNγ分泌情况。
取出细胞杀伤实验中(测试例5)收集的冻存于-20℃冰箱的上清，常温解冻，震荡混匀。使用无菌水溶解IFNγ标准品粉末，使用1640+10％ FBS培养基稀释标准品至需要的浓度。取出要检测IFNγ的试剂盒(CISBIO,62HIFNGPEG)，平衡试剂盒至常温，使用detection buffer稀释试剂盒中的两个检测抗体(20倍稀释)。取16μL细胞上清(10倍稀释用于IFNγ检测)及IFNγ标准品于96孔板中，加入4μL稀释好的对应的检测抗体；震荡混匀，1000rpm离心1分钟，常温孵育过夜。取出孵育过夜的样品，1000rpm离心1分钟，使用PHERAstar多功能酶标仪读取665nm和620nm的吸收值。用Graphpad Prism 8分析数据，结果见图5A。
结果显示，本披露的双特异性抗体释放IFNγ的水平非常低。
2.本披露的双特异性抗体的细胞因子IL-6释放水平
使用ELISA法检测在DLL3/CD3双抗在H1184细胞存在的况下，刺激PBMC中细胞因子IL-6分泌的情况。
取出细胞杀伤实验中(测试例5)收集的冻存于-20℃冰箱的上清，常温解冻，震荡混匀，样品使用样品稀释液稀释5倍备用。细胞因子检测前，取出IL-6ELISA试剂盒(欣博盛生物科技有限公司，EHC007.96)常温平衡，根据说明书分别稀释IL-6标准品和进行检测测量OD450值，用Graphpad Prism 8分析数据。结果见图5B。
结果显示，本披露的双特异性抗体释放IL6的水平非常低，说明本披露的双特异性抗体具有更好的安全性。
体内活性评价
测试例8.本披露的双特异性抗体在SHP77皮下移植瘤模型中的药效
本披露采用重度联合免疫缺陷NOG小鼠接种SHP-77细胞，接种后腹腔注射人PBMC，成瘤后分组给药。
NOG小鼠，雌性，体重15-17g左右，购自维通利华。SHP-77细胞来自ATCC。人PBMCs购自于妙顺(上海)生物科技有限公司，货号：ID#A10S115034。
将SHP-77细胞(4×106个/只小鼠)100μL接种于NOG小鼠右肋部皮下。接种当天，复苏冻存的hPBMC。接种第2天，每只小鼠腹腔注射hPBMC 5×106/100μL的细胞悬液。接种8天后，待肿瘤体积在～120mm3后去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为5组，每组8只，当天开始给等摩尔量的抗体，给药量如表32所示。通过腹腔注射相应等摩尔量的抗体，每5天给药1次，共给药3次，给药到第15天。每周测2次瘤体积，称体重，记录数据。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100％，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝100-T/C(％)。
表17.抗体在SHP77皮下移植瘤模型中的药效(给药后第15天)
结果显示，本披露的双特异性抗体可以显著抑制SHP-77移植瘤的生长。
测试例9.本披露的双特异性抗体在H1184皮下移植瘤模型中的药效
本披露采用重度联合免疫缺陷NOG小鼠接种H1184细胞，接种后腹腔注射人PBMC，成瘤后分组给药。
NOG小鼠，雌性，体重15-17g左右，购自维通利华。H1184细胞来自ATCC。人PBMCs购自于妙顺(上海)生物科技有限公司，货号：ID#A10S115034。
将H1184细胞(3.5×106个，含50％MatriGel)200μL/只接种于NOG小鼠右肋部皮下。接种第11天，复苏8支冻存hPBMC(ID#A10S115034)。接种第12天，每只小鼠腹腔注射人PBMC 5×106/100μL的细胞悬液。接种18天后，待肿瘤体积在～140mm3后去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为组，每组8只，当天开始给等摩尔量的抗体，给药量如表33所示。通过腹腔注射抗体，共给药4次，每周2次。每周测2次瘤体积，称体重，记录数据。
使用Excel统计软件记录数据：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT(每组动物数)计算；采用GraphPad Prism软件作图，采用Two-way ANOVA、One-way ANOVA、t-test对数据进行统计学分析。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100％，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝100-T/C(％)。
表18.抗体在H1184皮下移植瘤模型中的药效(给药后第15天)

结果显示，本披露的双特异性抗体可以显著抑制H1184移植瘤的生长。
二、制备实施例-特异性结合DLL3和CD3的双特异性抗体制剂
SEC分子排阻色谱法：
根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。
SEC％(SEC单体含量百分比)＝A单体/A总×100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和)。ΔSEC％＝稳定性实验前制剂的SEC％-稳定性实验后制剂的SEC％。
SEC测定用仪器：安捷伦HPLC 1260；
柱子：Waters，BioResolveTM SEC mAb2.5μm 7.8×300mm Column
NR-CE毛细管凝胶电泳：
将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。
NR-CE％(NR-CE主峰含量百分比)＝A主峰/A总×100％(A主峰为样品中主峰的峰面积，A总为所有峰面积之和)。ΔNR-CE％＝稳定性实验前制剂的NR-CE％-稳定性实验后制剂的NR-CE％。
CE测定用仪器：Beckman毛细管电泳仪，型号PA800 plus
iCIEF全柱成像毛细管等电聚焦电泳：
根据蛋白质等电点pI不同进行分离的技术。
iCIEF％(iCIEF中性峰含量百分比)＝A中性峰面积/A总面积×100％(A总面积为酸性峰、中性峰和碱性峰面积之和)。ΔiCIEF％＝稳定性实验前制剂的iCIEF％-稳定性实验后制剂的iCIEF％。
iCIEF测定用仪器：simple protein，型号Muarice。
IEC离子交换色谱：
以离子交换树脂或化学键合离子交换剂为固定相，利用被分离组分离子交换能力的差别或选择性系数的差别而实现分离的色谱方法。
IEC％(IEC中性峰含量百分比)＝A中性峰面积/A总面积×100％(A总面积为酸性峰、中性峰和碱性峰面积之和)。ΔIEC％＝稳定性实验前制剂的IEC％-稳定性实验后制剂的IEC％。
IEC测定用仪器：安捷伦HPLC 1260。
渗透压测定：
冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。
渗透压测定用仪器：罗泽Loser，型号OM815。
蛋白
以下实施例所采用的蛋白为特异性结合DLL3和CD3的双特异性抗体蛋白(hu6(D56E)-S107E-diabody，以下简称“蛋白”)。
融合蛋白的浓度测定用仪器：紫外可见分光光度计，型号：Nano Drop oneC，光程为1mm。
制剂实施例1.pH和缓冲体系筛选
用表19所示的缓冲体系制备70mg/mL蛋白、80mg/mL蔗糖、0.4mg/mL聚山梨酯80(PS80)的制剂。对样品进行强制降解研究(40℃放置4周)，以SEC和IEC为评价指标，进一步考察不同pH和缓冲体系对蛋白稳定性的影响。
结果见表19。SEC数据显示，ΔSEC在0.8％-3.2％范围内，其中His-HCl体系(pH4.5-5.5)和His-AA体系(pH5.0)组较优。ΔIEC数据显示，His-HCl(pH4.5-5.5)体系和CA体系(pH5.5)组较优。综合ΔSEC和ΔIEC数据，优选His-HCl体系(pH4.5-5.5)。
表19.pH和缓冲体系筛选结果
注：His-HCl代表组氨酸-盐酸组氨酸；SA代表琥珀酸-琥珀酸钠盐；CA代表枸橼酸-枸橼酸钠盐；His-AA代表组氨酸-醋酸组氨酸；40℃ W4：40℃放置4周，下同。
制剂实施例2.蛋白浓度筛选
用18mM的His-HCl缓冲剂(pH 5.0)制备0.1mg/mL二水合乙二胺四乙酸二钠(含二水，厂家：湖南尔康制药股份有限公司，后简称EDTA-2Na，CAS:6381-92-6)、80mg/mL蔗糖、0.4mg/mL PS80和表20所示的蛋白浓度的制剂。对样品进行强制降解研究(40℃放置4周)，以SEC和IEC为评价指标，考察不同蛋白浓度对蛋白稳定性的影响。
结果见表20。40℃放置4周，随着蛋白浓度提高，ΔSEC略有增加趋势，ΔIEC组间差异较小。
表20.蛋白浓度筛选结果
制剂实施例3.EDTA-2Na浓度筛选
用18mM的His-HCl缓冲剂(pH 5.0)制备100mg/mL蛋白、80mg/mL蔗糖、0.4mg/mL PS80和表21所示的不同浓度EDTA-2Na的制剂。对样品进行强制降解研究(40℃放置4周)，以SEC、NR-CE和IEC为评价指标，考察不同EDTA-2Na浓度对制剂稳定性的影响。
结果见表21。40℃放置4周，ΔSEC在0.7％-1.4％范围内，含0.1mg/mL EDTA-2Na的制剂单体纯度略优于其他组。ΔNR-CE和ΔIEC组间无显著差异。
表21.EDTA-2Na浓度筛选结果
制剂实施例4.His-HCl缓冲体系离子浓度筛选
用表22所示的不同离子浓度的His-HCl缓冲剂(pH 5.0)制备100mg/mL蛋白、0.1mg/mL EDTA-2Na、80mg/mL蔗糖和0.4mg/mL PS80的制剂。对样品进行强制降解研究(40℃放置4周)，以SEC、NR-CE和IEC为评价指标，考察不同组氨酸离子浓度对蛋白稳定性的影响。
结果见表22。40℃放置4周，ΔSEC、ΔNR-CE和ΔIEC组间无显著差异。
表22.His-HCl缓冲体系浓度筛选结果
制剂实施例5.糖浓度筛选
用18mM的His-HCl缓冲剂(pH 5.0)制备100mg/mL蛋白、0.1mg/mL EDTA-2Na、0.4mg/mL PS80和表23所示的不同浓度蔗糖的制剂。对样品进行强制降解研究(40℃放置4周)，以SEC、NR-CE和IEC为评价指标，考察不同浓度蔗糖对制剂稳定性的影响。
结果见表23。ΔSEC在0.7％-1.5％范围内，含80mg/mL蔗糖的制剂单体纯度略优于其他组。ΔNR-CE和ΔIEC组间无显著差异。
表23.蔗糖浓度筛选结果
糖作为生物药常用辅料，本项目采用蔗糖作为渗透压调节剂及稳定剂进行处方筛选，可满足制剂要求。蔗糖浓度为80mg/mL时，实际测定制剂的渗透压为292mOsm，接近等渗。因此，优选蔗糖浓度为80mg/mL。
制剂实施例6.表面活性剂种类筛选
用18mM的His-HCl缓冲剂(pH 5.5)制备70mg/mL蛋白、80mg/mL蔗糖和表24所示不同种类表面活性剂的制剂，包括PS80和泊洛沙姆188(P188)。对样品进行强制降解研究(40℃放置4周)，以外观、SEC、NR-CE和iCIEF为评价指标，考察不同种类表面活性剂对蛋白稳定性的影响。
结果见表24。外观、ΔSEC、ΔNR-CE和ΔiCIEF数据显示，40℃放置4周后，含不同种类表面活性剂的制剂外观和纯度项无显著差异。
表24.表面活性剂浓度筛选结果
制剂实施例7.表面活性剂浓度筛选
用18mM的His-HCl缓冲剂(pH 5.0)制备100mg/mL蛋白、0.1mg/mL EDTA-2Na、80mg/mL蔗糖和表25所示不同浓度PS80的制剂。对样品进行强制降解研究(40℃放置4周)，以外观、SEC、NR-CE和IEC为评价指标，考察不同浓度表面活性剂对蛋白稳定性的影响。
结果见表25。外观、ΔSEC、ΔNR-CE和ΔIEC数据显示，40℃放置4周后，含不同浓度PS80的制剂外观和纯度项无显著差异。
表25.表面活性剂浓度筛选结果
制剂实施例8.处方确认
用18mM的His-HCl缓冲剂(pH 5.0)制备150mg/mL蛋白、0.1mg/mL EDTA-2Na、80mg/mL蔗糖和0.4mg/mL PS80的制剂。对样品进行长期稳定性研究(5℃放置6个月)，以SEC和IEC为评价指标，考察该处方的蛋白稳定性。
结果见表26。5℃放置6个月，与第0天相比，SEC和IEC均无显著变化，因此在该蛋白浓度条件下，蛋白稳定性良好。
表26.蛋白浓度150mg/mL处方长期稳定性结果
制剂实施例9.处方确认
用18mM His-HCl缓冲剂(pH 5.0)制备100mg/mL蛋白、0.1mg/mL EDTA-2Na、0.4mg/mL PS80和80mg/mL蔗糖的制剂。对样品进行长期稳定性研究，以SEC、NR-CE、IEC为评价指标，考察长期稳定性。
结果见表27。SEC、NR-CE和IEC数据显示，5℃放置12个月后制剂纯度项无显著变化。
表27.长期稳定性结果
注：5℃，M3/M12：5℃放置3个月/12个月。
制剂实施例10.处方确认
用18mM His-HCl缓冲剂(pH 5.0)制备5mg/mL蛋白、0.1mg/mL EDTA-2Na、0.4mg/mL PS80和80mg/mL蔗糖的制剂。对样品进行长期稳定性研究，以SEC、IEC为评价指标，考察长期稳定性。
结果见表28。SEC和IEC数据显示，5℃放置6个月后制剂纯度项无显著变化。
表28.长期稳定性结果
注：5℃，M1/M6：5℃放置1个月/6个月。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本披露的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (22)

  1. 一种药物组合物，包含特异性结合DLL3和CD3的双特异性抗体和缓冲剂，其中：
    所述特异性结合DLL3和CD3的双特异性抗体包含一条具有式I所示结构的第一链和一条具有式II所示结构的第二链，
    式I：[DLL3-VL]-[连接子1]-[CD3-VH]-[连接子2]-[Fc1]，
    式II：[CD3-VL]-[连接子3]-[DLL3-VH]-[连接子2]-[Fc2]，
    所述DLL3-VH包含SEQ ID NO：14的氨基酸序列的DLL3-HCDR1，包含SEQ ID NO：15的氨基酸序列的DLL3-HCDR2和包含SEQ ID NO：16的氨基酸序列的DLL3-HCDR3；和所述DLL3-VL具有：包含SEQ ID NO：17的氨基酸序列的DLL3-LCDR1，包含SEQ ID NO：50的氨基酸序列的DLL3-LCDR2，和包含SEQ ID NO：19的氨基酸序列的DLL3-LCDR3；和
    所述CD3-VH包含SEQ ID NO：56的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：57的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：58的氨基酸序列的CD3-HCDR3；和所述CD3-VL具有：包含SEQ ID NO：59的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：60的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：61的氨基酸序列的CD3-LCDR3；
    其中：式I和式II所示的结构是从N端至C端排列的；所述连接子1、连接子2和连接子3是相同或不同的肽连接子；所述Fc1和所述Fc2是能够以杵臼技术相互缔合的Fc区结构序列；
    所述缓冲剂为组氨酸盐缓冲剂、醋酸盐缓冲剂、枸橼酸盐缓冲剂、琥珀酸盐缓冲剂或磷酸盐缓冲剂；
    优选地，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂、组氨酸-醋酸组氨酸缓冲剂或枸橼酸-枸橼酸钠盐缓冲剂；
    更优选地，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂。
  2. 根据权利要求1所述的药物组合物，其中所述药物组合物的pH为4.5至6.0；优选地，所述药物组合物的pH为4.5至5.5；更优选地，所述药物组合物的pH为4.8至5.2；最优选地，所述药物组合物的pH为约5.0。
  3. 根据权利要求1或2所述的药物组合物，其中所述特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至250mg/mL；
    优选地，所述特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至150mg/mL；
    进一步优选地，所述特异性结合DLL3和CD3的双特异性抗体的浓度为70mg/mL至150mg/mL；
    更优选地，所述特异性结合DLL3和CD3的双特异性抗体的浓度为80mg/mL至120mg/mL；
    最优选地，所述特异性结合DLL3和CD3的双特异性抗体的浓度为约100mg/mL。
  4. 根据权利要求1或2所述的药物组合物，其中所述特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至20mg/mL；
    优选地，所述特异性结合DLL3和CD3的双特异性抗体的浓度为1mg/mL至10mg/mL；
    更优选地，所述特异性结合DLL3和CD3的双特异性抗体的浓度为4mg/mL至6mg/mL；
    最优选地，所述特异性结合DLL3和CD3的双特异性抗体的浓度为约5mg/mL。
  5. 根据权利要求1至4任一项所述的药物组合物，其中所述药物组合物包含表面活性剂；优选地，所述表面活性剂为聚山梨酯或泊洛沙姆；更优选地，所述表面活性剂为聚山梨酯80或泊洛沙姆188；最优选地，所述表面活性剂为聚山梨酯80。
  6. 根据权利要求5所述的药物组合物，其中所述聚山梨酯80的浓度为0.01mg/mL至1.0mg/mL；优选地，所述聚山梨酯80的浓度为0.1mg/mL至1.0mg/mL；进一步优选地，所述聚山梨酯80的浓度为0.2mg/mL至0.6mg/mL；更优选地，所述聚山梨酯80的浓度为0.3mg/mL至0.5mg/mL；最优选地，所述聚山梨酯80的浓度为约0.4mg/mL。
  7. 根据权利要求5所述的药物组合物，其中所述泊洛沙姆188的浓度为0.5mg/mL至5.0mg/mL；优选地，所述泊洛沙姆188的浓度为1mg/mL至3mg/mL；更优选地，所述泊洛沙姆188的浓度为1.5mg/mL至2.5mg/mL；最优选地，所述泊洛沙姆188的浓度为约2.0mg/mL。
  8. 根据权利要求1至7任一项所述的药物组合物，其中所述药物组合物包含糖；优选地，所述糖为蔗糖、海藻糖、甘露醇或山梨糖醇；更优选地，所述糖为蔗糖。
  9. 根据权利要求8所述的药物组合物，其中所述糖的浓度为10mg/mL至120mg/mL；优选地，所述糖的浓度为30mg/mL至100mg/mL；更优选地，所述糖的浓度为64mg/mL至96mg/mL；最优选地，所述糖的浓度为约80mg/mL。
  10. 根据权利要求1至9任一项所述的药物组合物，其中所述缓冲剂的浓度为5mM至100mM；优选地，所述缓冲剂的浓度为10mM至50mM；进一步优选地，所述缓冲剂的浓度为10mM至30mM；更优选地，所述缓冲剂的浓度为15mM至25mM；最优选地，所述缓冲剂的浓度为约18mM。
  11. 根据权利要求1至10任一项所述的药物组合物，其中所述药物组合物还包含辅料；优选地，所述辅料为二水合乙二胺四乙酸二钠、DTPA、盐酸精氨酸、甘氨酸、甲硫氨酸、脯氨酸、组氨酸、苯丙氨酸、谷氨酸、天冬氨酸、氯化钠或氯化钙；更优选地，所述辅料为二水合乙二胺四乙酸二钠。
  12. 根据权利要求11所述的药物组合物，其中所述辅料的浓度为0.01mg/mL至1mg/mL；优选地，所述辅料的浓度为0.01mg/mL至0.5mg/mL；更优选地，所述辅料的浓度为0.08mg/mL至0.12mg/mL；最优选地，所述辅料的浓度为约0.1mg/mL。
  13. 根据权利要求1至12任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中：
    所述DLL3-VH包含SEQ ID NO：33的氨基酸序列，和所述DLL3-VL包含SEQ ID NO：42的氨基酸序列；和
    所述CD3-VH包含SEQ ID NO：62的氨基酸序列，和所述CD3-VL包含SEQ ID NO：63的氨基酸序列。
  14. 根据权利要求1至13任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构；
    优选地，所述Fc1的氨基酸序列如SEQ ID NO：66所示；和所述Fc2的氨基酸序列如SEQ ID NO：67所示。
  15. 根据权利要求1至14任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体中所述的连接子1、连接子2和连接子3相同或不同，各自独立地选自SEQ ID NO：68、SEQ ID NO：69和SEQ ID NO：70。
  16. 根据权利要求1至15任一项所述的药物组合物，其中特异性结合DLL3和CD3的双特异性抗体，其中
    式I的氨基酸序列如SEQ ID NO：71所示，和式II的氨基酸序列如SEQ ID NO：72所示。
  17. 根据权利要求1所述的药物组合物，其包含如下组分：
    (a)1mg/mL至250mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.01mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
    (c)10mg/mL至120mg/mL的糖，
    (d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
    (e)5mM至100mM的缓冲剂，所述药物组合物的pH为4.5至6.0；
    优选地，所述药物组合物包含如下组分：
    (a)1mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.1mg/mL至1.0mg/mL的聚山梨酯80或1mg/mL至3mg/mL泊洛沙姆188，
    (c)30mg/mL至100mg/mL的蔗糖，
    (d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
    (e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂、组氨酸-醋酸组氨酸缓冲剂或枸橼酸-枸橼酸钠盐缓冲剂，所述药物组合物的pH为4.5至5.5；
    进一步优选地，所述药物组合物包含如下组分：
    (a)70mg/mL至150mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.2mg/mL至0.6mg/mL的聚山梨酯80或1mg/mL至3mg/mL泊洛沙姆188，
    (c)64mg/mL至96mg/mL的蔗糖，
    (d)0.01mg/mL至0.5mg/mL的二水合乙二胺四乙酸二钠，和
    (e)10mM至30mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为4.5至5.5；
    更优选地，所述药物组合物包含如下组分：
    (a)80mg/mL至120mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.3mg/mL至0.5mg/mL的聚山梨酯80或1.5mg/mL至2.5mg/mL泊洛沙姆188，
    (c)64mg/mL至96mg/mL的蔗糖，
    (d)0.08mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠，和
    (e)15mM至25mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为4.8至5.2：
    最优选地，所述药物组合物包含如下组分：
    (a)约100mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b约0.4mg/mL的聚山梨酯80，
    (c)约80mg/mL的蔗糖，
    (d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
    (e)约18mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为约5.0。
  18. 根据权利要求1所述的药物组合物，其包含如下组分：
    (a)1mg/mL至20mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.01mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
    (c)10mg/mL至120mg/mL的糖，
    (d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
    (e)5mM至100mM的缓冲剂，所述药物组合物的pH为4.5至6.0；
    优选地，所述药物组合物包含如下组分：
    (a)1mg/mL至10mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.1mg/mL至1.0mg/mL的聚山梨酯80或0.5mg/mL至5mg/mL泊洛沙姆188，
    (c)30mg/mL至100mg/mL的蔗糖，
    (d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
    (e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂、组氨酸-醋酸组氨酸缓冲剂或枸橼酸-枸橼酸钠盐缓冲剂，所述药物组合物的pH为4.5至5.5；
    进一步优选地，所述药物组合物包含如下组分：
    (a)1mg/mL至10mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.1mg/mL至1.0mg/mL的聚山梨酯80，
    (c)30mg/mL至100mg/mL的蔗糖，
    (d)0.01mg/mL至1mg/mL的二水合乙二胺四乙酸二钠，和
    (e)10mM至50mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为4.5至5.5；
    更优选地，所述药物组合物包含如下组分：
    (a)4mg/mL至6mg/mL的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)0.3mg/mL至0.5mg/mL的聚山梨酯80，
    (c)64mg/mL至96mg/mL的蔗糖，
    (d)0.08mg/mL至0.12mg/mL的二水合乙二胺四乙酸二钠，和
    (e)15mM至25mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为4.8至5.2；
    最优选地，所述药物组合物包含如下组分：
    (a)约5mg/m的所述特异性结合DLL3和CD3的双特异性抗体，
    (b)约0.4mg/mL的聚山梨酯80，
    (c)约80mg/mL的蔗糖，
    (d)约0.1mg/mL的二水合乙二胺四乙酸二钠，和
    (e)约18mM的组氨酸-盐酸组氨酸缓冲剂，所述药物组合物的pH为约5.0。
  19. 根据权利要求1至18任一项所述的药物组合物，其为皮下注射制剂、静脉注射制剂、腹腔注射制剂或肌肉注射制剂；优选为皮下注射制剂。
  20. 一种制备冻干制剂的方法，其中包括将权利要求1至18任一项所述的药物组合物进行冷冻干燥的步骤。
  21. 一种冻干制剂，所述制剂通过权利要求20所述的方法获得的。
  22. 权利要求1至19任一项所述的药物组合物，或权利要求21所述的冻干制剂在制备用于治疗肿瘤或癌症的药物中的用途；优选地，其中所述肿瘤或癌症选自：
    肺癌、小细胞肺癌、大细胞肺癌、头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、咽鳞癌、口腔鳞癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、大细胞肺癌、三阴性乳腺癌和淋巴瘤。
PCT/CN2024/140832 2023-12-22 2024-12-20 一种包含特异性结合dll3和cd3的双特异性抗体的药物组合物 Pending WO2025131024A1 (zh)

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