WO2024153224A1 - 一种含艾日布林衍生物药物偶联物的药物组合物 - Google Patents

一种含艾日布林衍生物药物偶联物的药物组合物 Download PDF

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Publication number
WO2024153224A1
WO2024153224A1 PCT/CN2024/073229 CN2024073229W WO2024153224A1 WO 2024153224 A1 WO2024153224 A1 WO 2024153224A1 CN 2024073229 W CN2024073229 W CN 2024073229W WO 2024153224 A1 WO2024153224 A1 WO 2024153224A1
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Prior art keywords
pharmaceutical composition
cancer
drug conjugate
buffer
concentration
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English (en)
French (fr)
Inventor
吴婷婷
杜敏
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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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Priority to EP24744394.8A priority Critical patent/EP4653012A1/en
Priority to CN202480005918.7A priority patent/CN120417925A/zh
Priority to KR1020257024906A priority patent/KR20250129056A/ko
Priority to JP2025540970A priority patent/JP2026504859A/ja
Publication of WO2024153224A1 publication Critical patent/WO2024153224A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition containing an eribulin derivative drug conjugate.
  • Antibody drug conjugates connect monoclonal antibodies or antibody fragments to biologically active drugs through stable chemical linker compounds, making full use of the specificity of antibodies binding to surface antigens of normal cells and tumor cells and the high efficiency of drugs, while avoiding the defects of the former's low efficacy and the latter's excessive toxic side effects. This means that compared with traditional chemotherapy drugs, antibody drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells.
  • Microtubules are powerful filamentous cytoskeletal proteins associated with various cell functions including intracellular migration and transport, cell signaling, and maintaining cell shape. Microtubules also play a key role in mitotic cell division by forming the mitotic spindle required for chromosome division into two daughter cells. Most of the biological functions of microtubules in all cells are regulated by their polymerization dynamics, which are reversibly and non-covalently added to the two ends of the microtubules by ⁇ and ⁇ tubulin dimers. This dynamic behavior and the resulting control of microtubule length are indispensable for the proper function of the mitotic spindle.
  • tubulin inhibitors and antibody drug conjugates thereof are expected to become a class of promising drugs for the treatment of cancer.
  • ADCs have a more complex and heterogeneous structure than antibodies, thus posing greater challenges to ADC formulation for therapeutic purposes.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody drug conjugate and a buffer, wherein the antibody drug conjugate has a structure as shown in Formula I:
  • Ab is Pertuzumab
  • n 1 to 10;
  • the buffer is selected from the group consisting of citrate buffer, histidine buffer and succinate buffer.
  • the buffer is selected from the group consisting of histidine-histidine hydrochloride, citric acid-sodium citrate, and succinic acid-sodium succinate.
  • the range of drug loading (n) can be the average number of cytotoxic drugs bound to each Pertuzumab antibody, non-limiting examples include the average number of cytotoxic drugs bound to each antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and any range between these point values.
  • it can be 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 3.5-4.7, 5-6, 5-7, 5-8 and 6-8.
  • the drug loading (n) can be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • n is a decimal or an integer. In some embodiments, n is 1 to 8, or 3 to 5.
  • the pH of the pharmaceutical composition is 3.5 to 5.5, non-limiting examples include 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, and any range between these points.
  • the pH is 3.5 to 5.0, or the pH is 3.7 to 4.7.
  • the buffer concentration in the pharmaceutical composition is 5 mM to 50 mM, non-limiting examples include 5 mM, 10 mM, 12 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM and any range between these points; in some embodiments, the buffer concentration is 10 mM to 50 mM; in some embodiments, the buffer concentration is 20 mM to 40 mM; in some embodiments, the buffer concentration is 30 mM.
  • the pharmaceutical composition further comprises a surfactant. It can be selected from polysorbate, poloxamer, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauryl amide Propyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl
  • the concentration of the surfactant in the pharmaceutical composition is 0.01 mg/mL to 1.0 mg/mL, or 0.1 mg/mL to 0.8 mg/mL, or 0.3 mg/mL to 0.8 mg/mL. In some embodiments, the concentration of the surfactant is 0.6 mg/mL.
  • Non-limiting examples include 0.02 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.15 mg/mL, 0.2 mg/mL, 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL, 0.45 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, and any range between these points.
  • the aforementioned pharmaceutical composition further comprises sugar.
  • the "sugar” disclosed herein comprises conventional compositions ( CH2O ) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like.
  • the sugar may be selected from glucose, sucrose, trehalose, ⁇ , ⁇ -trehalose dihydrate, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like.
  • the sugar is sucrose.
  • the concentration of sugar in the aforementioned pharmaceutical composition is 25 mg/mL to 80 mg/mL, or 30 mg/mL to 50 mg/mL, non-limiting examples include 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL and any range between these point values. In some embodiments, the concentration is 40 mg/mL.
  • the aforementioned pharmaceutical composition further comprises an amino acid, such as glycine.
  • the concentration of the amino acid in the aforementioned pharmaceutical composition is 6 mg/mL to 15 mg/mL, for example, 7 mg/mL to 11 mg/mL, or 7 mg/mL to 10 mg/mL, non-limiting examples include 6 mg/mL, 6.5 mg/mL, 7 mg/mL, 7.2 mg/mL, 7.6 mg/mL, 7.8 mg/mL, 8 mg/mL, 8.5 mg/mL, 9 mg/mL, 10 mg/mL, 10.2 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL and any range between these point values. In some embodiments, the concentration is 9 mg/mL.
  • the concentration of the antibody drug conjugate in the pharmaceutical composition is 1 mg/mL to 100 mg/mL based on protein concentration, and non-limiting examples include 1 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL, 21 mg/mL, 22 mg/mL, 23 mg/mL, In some embodiments, the concentration of the antibody drug conjugate is 10 mg/mL to 30 mg/mL, for example 20 mg/mL, based on protein concentration.
  • non-limiting examples include 20.1 mg/mL, 20.2 mg/mL, 20.3 mg/mL, 20.4 mg/mL, 20.5 mg/mL, 20.6 mg/mL, 20.7 mg/mL, 20.8 mg/mL, 20.81 mg/mL, 20.82 mg/mL, 20.83 mg/mL, 20.84 mg/mL, 20.85 mg/mL, 20.86 mg/mL, 20.87 mg/mL, 20.88 mg/mL, 20.89 mg/mL, 21.
  • the protein concentration refers to the concentration of the antibody portion in the antibody drug conjugate.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • any of the foregoing pharmaceutical compositions is a liquid preparation.
  • the liquid preparation or the reconstituted preparation disclosed herein has good stability.
  • a stable liquid preparation includes a liquid preparation that exhibits desired characteristics after being stored at a temperature of 40° C. for 1 month.
  • the present disclosure also provides a lyophilized formulation containing an antibody drug conjugate, wherein the formulation can form the pharmaceutical composition as described above after reconstitution.
  • the present disclosure also provides a method for preparing a lyophilized formulation containing an antibody drug conjugate, which comprises the step of freeze-drying the pharmaceutical composition as described above.
  • the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, at least 28 days, or at least 30 days.
  • the present disclosure also provides a lyophilized preparation comprising an antibody drug conjugate, wherein the lyophilized preparation is obtained by freeze-drying the pharmaceutical composition of the antibody drug conjugate as described above.
  • the present disclosure also provides a reconstituted solution containing an antibody drug conjugate, wherein the reconstituted solution is prepared by reconstituted the lyophilized preparation as described above.
  • the present disclosure also provides a method for preparing the above-mentioned reconstituted solution, which comprises the step of reconstituted the above-mentioned lyophilized preparation, and the solution used for reconstitution is selected from but not limited to water for injection, physiological saline or glucose solution.
  • the present disclosure also provides a product, which includes a container, wherein the pharmaceutical composition, lyophilized preparation or reconstituted solution as described above is contained in the container.
  • the container is a neutral borosilicate glass tube injection bottle.
  • the present disclosure also provides use of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or product in the preparation of a drug for treating or preventing tumors.
  • the present disclosure also provides a method for treating a disease, comprising administering the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or preparation to a patient in need thereof.
  • the present disclosure also provides the aforementioned pharmaceutical composition, or lyophilized preparation, or reconstituted solution, or product as a medicine for treating a disease.
  • the present disclosure also provides the aforementioned pharmaceutical composition as a drug, or a lyophilized preparation, or a reconstituted solution, or Products used to treat tumors.
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition, or lyophilized preparation, or reconstituted solution, or product as a drug in the preparation of a drug for treating tumors.
  • the tumor is a cancer associated with expression of domain II of HER2.
  • the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.
  • variable region sequences of the heavy and light chains of Pertuzumab are the variable region sequences of the heavy and light chains of Pertuzumab:
  • the present disclosure provides a pharmaceutical composition that is more conducive to production and administration and has stable performance.
  • the undesirable instability may include any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine, dissociation of toxins, etc.
  • the pharmaceutical composition described in the present disclosure comprises an antibody drug conjugate and a buffer.
  • ADCs Antibody-drug conjugates are antibodies that are linked to biologically active cytotoxins or small molecule drugs with cell-killing activity through a linker unit.
  • Drug loading or “drug loading” is also called the drug-to-antibody ratio (DAR), which is the average number of drugs coupled to each antibody in the ADC. It can be, for example, in the range of 1 to 10 drugs coupled to each antibody, and in certain embodiments, in the range of 1 to 8 drugs coupled to each antibody, preferably from 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8 and 6-8. Exemplarily, the drug loading can be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the ADC formula of the present disclosure includes a collection of antibodies coupled to drugs within the aforementioned certain range. In the embodiments of the present disclosure, the drug loading can be expressed as n. The drug loading can be determined by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC.
  • linker unit or “connection fragment” or “connection unit” refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.
  • Linkers including extenders, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US20050238649A1.
  • the linker can be a "cleavable linker" that facilitates release of the drug in the cell.
  • an acid-labile linker e.g., hydrazone
  • a protease-sensitive linker e.g., peptidase-sensitive
  • a photolabile linker e.g., peptidase-sensitive linker
  • a dimethyl linker e.g., a disulfide-containing linker
  • drug linker fragment or “drug-linker fragment” refers to a fragment formed by connecting a drug to a linker unit.
  • the segment can be connected to the antibody through the other end of the linker unit.
  • cytotoxic drugs can be controlled by the following non-limiting methods, including:
  • antibody described in the present disclosure is used in the broadest sense and covers various antibody structures, including but not limited to full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • a natural complete antibody is composed of a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region.
  • Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies.
  • the culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer.
  • Non-specifically bound components are washed away.
  • the bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected.
  • the antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product must be immediately frozen, such as at -70°C, or freeze-dried.
  • 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 succinic acid 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 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 disodium hydrogen phosphate-sodium dihydrogen 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.
  • “Pharmaceutical composition” means a mixture containing one or more antibody drug conjugates described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the antibody active ingredient, promote administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • composition and “formulation” are not mutually exclusive.
  • compositions described in the present disclosure are in the form of solutions, and unless otherwise specified, the solvent therein is water.
  • “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 terms “about” and “approximately” refer to values within an acceptable error range for a specific value determined by a person of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation in each practice in the art. Alternatively, “about” or “substantially including” can mean a range of up to 20%. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about” or “substantially including” should be assumed to be within an acceptable error range for the specific value.
  • the numerical values in this disclosure are instrumental measurements or calculated values after instrumental measurements, and there is a certain degree of error. Generally speaking, plus or minus 10% is within the reasonable error range. Of course, the context in which the numerical value is used needs to be considered.
  • the total impurity content which is a numerical value with an error change of no more than plus or minus 10% after measurement, can be plus or minus 9%, plus or minus 8%, plus or minus 7%, plus or minus 6%, plus or minus 5%, plus or minus 4%, plus or minus 3%, plus or minus 2% or plus or minus 1%, preferably plus or minus 5%.
  • the pharmaceutical composition disclosed herein can achieve a stable effect: a pharmaceutical composition in which the antibody drug conjugate substantially retains its physical stability and/or chemical stability and/or biological activity after storage; preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage.
  • the storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition.
  • analytical techniques for measuring the stability of proteins or antibody drug conjugates which can measure the stability after storage at a selected temperature for a selected period of time.
  • a stable formulation is one in which no significant change is observed when stored at a refrigerated temperature (2-8°C) for at least 3 months, preferably 6 months, and more preferably 1 year.
  • 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, 2 months, or 3 months.
  • a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after being stored at a temperature of 40°C or more. The desired characteristics are exhibited after being stored at a temperature of 10 days, 20 days, 1 month, etc.
  • Typical examples of stability Aggregation or degradation of antibody monomers is usually not more than about 10%, preferably not more than about 5%, as measured by SEC-HPLC.
  • the formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white.
  • concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation have no more than ⁇ 10% variation. A reduction of no more than about 10%, preferably no more than about 5%, is usually observed. Aggregations of no more than about 10%, preferably no more than about 5%, are usually formed.
  • the antibody drug conjugate "retains its physical stability" in the 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 evaluated by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An antibody drug conjugate "retains its chemical stability" in a pharmaceutical formulation if it does not show 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 drug conjugate "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.
  • Substituted means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.
  • carrier is used for the drugs disclosed herein and refers to a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ.
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, and preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as excellent drug carriers.
  • administer when applied to animals, humans, experimental subjects, cells, tissues, organs or biological processes
  • administer refers to, for example, contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid.
  • administer may refer to, for example, treatment, pharmacokinetics, diagnosis, research and experimental procedures.
  • Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell.
  • administerister and “treat” also mean in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnosis, a combination composition or by another cell.
  • Treatment when applied to humans, veterinary medicine or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of therapeutic agent effective to alleviate any specific disease symptom may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by physicians or other professional health care personnel to evaluate the severity or progression of the symptom.
  • the embodiments of the present disclosure e.g., treatment methods or products
  • may not be effective in alleviating every target disease symptom they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's general health, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
  • Dislacement refers to the replacement of the solvent system for dissolving the antibody protein or antibody drug conjugate, for example, using the buffer system of the stable preparation to replace the high salt or hypertonic solvent system containing the antibody protein or antibody drug conjugate by physical operation, so that the antibody protein or antibody drug conjugate is present in the stable preparation.
  • the so-called physical operation includes but is not limited to ultrafiltration, dialysis or centrifugation followed by re-dissolution.
  • Pertuzumab refers to an antibody comprising the light chain and heavy chain variable region amino acid sequences in SEQ ID No. 1 and 2, respectively. If Pertuzumab is a complete antibody, it preferably comprises the light chain and heavy chain amino acid sequences in SEQ ID No. 3 and 4, respectively.
  • Figure 1 is a graph showing the changes in tumor volume in mice with N87/16-8 subcutaneous transplanted tumor model.
  • FIG. 2 is a graph showing changes in body weight of mice in the N87/16-8 subcutaneous transplanted tumor model.
  • FIG3 is a graph showing the changes in tumor volume in mice with JIMIT-1 subcutaneous transplanted tumor model.
  • FIG. 4 is a graph showing the weight changes of mice in the JIMIT-1 subcutaneous transplanted tumor model.
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • ⁇ NMR shifts ( ⁇ ) are given in units of 10 -6 (ppm).
  • NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD) as the measuring solvent, and tetramethylsilane (TMS) as the internal standard.
  • DMSO-d 6 deuterated dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS tetramethylsilane
  • MS was determined using an Agilent 1200/1290 DAD-6110/6120 Quadrupole MS LC-MS/MS instrument (manufacturer: Agilent, MS model: 6110/6120 Quadrupole MS).
  • HPLC High performance liquid chromatography
  • Chiral HPLC analysis was performed using an Agilent 1260 DAD high performance liquid chromatograph.
  • HPLC preparation was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.
  • the CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate.
  • the silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.15mm to 0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm to 0.5mm.
  • Silica gel column chromatography generally uses Yantai Huanghai Silica Gel 200 to 300 mesh silica gel as the carrier.
  • the known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals and other companies.
  • the reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.
  • Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1L.
  • Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.
  • the pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Clear Blue QL-500 hydrogen generator or a HC2-SS hydrogenator.
  • the hydrogenation reaction is usually carried out by evacuating the vacuum, filling with hydrogen, and repeating the operation three times.
  • Microwave reactions were performed using a CEM Discover-S 908860 microwave reactor.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, 20°C to 30°C.
  • the eluent system of column chromatography and the developing solvent system of thin layer chromatography used for purifying compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and ethyl acetate system.
  • A dichloromethane and isopropanol system
  • B dichloromethane and methanol system
  • C petroleum ether and ethyl acetate system.
  • the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.
  • the antibody drug conjugate as shown in Formula I described herein can be prepared according to the method of PCT/CN2022/107479.
  • compound 1a (eribulin, prepared according to ZL201010236637.2) (72.91 mg, 0.1 mmol) was dissolved in 10 mL of tetrahydrofuran, Fmoc-OSu (fluorenylmethoxycarbonyl succinimide, 41 mg, 0.12 mmol) was added, and then stirred at room temperature until the reaction was complete. The crude product was concentrated under reduced pressure and used directly in the next step.
  • the crude compound 1b obtained in the previous step was dissolved in 10 mL of anhydrous ether, and silver oxide (34.8 mg, 0.15 mmol) was added, followed by iodomethane (28.4 mg, 0.2 mmol). The mixture was stirred at room temperature until the reaction was complete, filtered, and then concentrated under reduced pressure to obtain a crude product, which was directly subjected to the next step of reaction.
  • the crude compound 1c obtained in the previous step was dissolved in 10 mL of tetrahydrofuran, and 2 mL of diethylamine was added. The mixture was stirred at room temperature until the reaction was complete, and the crude product was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane/ethyl acetate/petroleum ether) to obtain 3 mg of the target product compound 1.
  • the reaction solution was cooled to 25°C in a water bath.
  • Dissolve compound L-1 (6.865 g, 4.94 mmol) in 0.425 L DMSO to generate a DMSO solution of compound L-1.
  • Pre-add 0.348 L DMSO to the intermediate I solution then add the DMSO solution of compound L-1 to the intermediate I solution pre-added with DMSO, and stir in a water bath at 12°C.
  • Add 2 M acetic acid dropwise to adjust the pH of the reaction solution to 4.2.
  • Test Example 1 Inhibitory effect on proliferation of in vitro cultured human breast cancer BT-474 and human gastric cancer NCI-N87, NCI-N87/16-8, NCI-N87/8-2 cells
  • NCI-N87 and BT-474 cells were purchased from American Type Culture Collection (ATCC). T-DM1-resistant NCI-N87/16-8 and NCI-N87/8-2 cells induced by long-term exposure of NCI-N87 to T-DM1 were constructed by our laboratory. The cells were cultured in RPMI 1640/DMEM (1:1) medium containing 10% fetal bovine serum (FBS).
  • ATCC American Type Culture Collection
  • FBS fetal bovine serum
  • RPMI 1640 and DMEM were purchased from Gibco BRL; FBS was purchased from Gibco; and sulforhodamine B (SRB) was purchased from Sigma.
  • the ELISA reader Synergy H4 was purchased from BioTek.
  • a certain number of cells in logarithmic growth phase were inoculated into 96-well culture plates. After 24 hours of adherent growth, different concentrations of drugs (10000, 3000, 1000, 300, 100, 30, 10, 3, 1 ng/mL) were added. After 120 hours of drug treatment, the cells were fixed with trichloroacetic acid. Then SRB solution was used for staining; finally, Tris solution was added to dissolve SRB, and the OD value was measured at a wavelength of 510 nm by an ELISA reader. The cell growth inhibition rate was calculated using the following formula:
  • Inhibition rate (OD value of control well - OD value of drug administration well) / OD value of control well ⁇ 100%
  • the half inhibition concentration IC 50 was calculated using GraphPad Prism 7 software.
  • Test Example 2 In vitro cytotoxic activity screening of compound 1
  • This experiment uses CTG to detect ATP content, reflecting the survival of tumor cells.
  • SKBR3 tumor cells HER2+, ATCC, catalog number HTB-30
  • MDA-MB-468 HER2-, ATCC, catalog number HTB-132
  • A549 human non-small cell lung cancer cells, ATCC, catalog number CCL-185
  • CTG assay Cell Titer-GloTM, luminescent cell viability assay, Promega: The cell plates were removed on the 3rd and 5th days, and equilibrated to room temperature. 90ul CTG was added to each well, and the cells were reacted at room temperature in the dark for 10 minutes. The luminescence value was read by a microplate reader and the IC 50 was calculated.
  • Beagles were used as test animals, and the drug concentrations in the plasma of beagles at different times after intravenous injection of compound 1 and eribulin were determined by LC/MS/MS.
  • the pharmacokinetic behavior of the disclosed compound in dogs was studied, and its pharmacokinetic characteristics were evaluated.
  • Compound 1 was weighed, and 5% volume of DMSO, 20% PG and 20% PEG400 were added to dissolve it, and then 55% physiological saline was added to prepare a 0.25 mg/ml colorless clear solution.
  • Eribulin was weighed, and 5% volume of DMSO, 20% PG and 20% PEG400 were added to dissolve it, and then 55% physiological saline was added to prepare a 0.25 mg/ml colorless clear solution.
  • a group of dogs were intravenously administered with compound 1, the dosage was 0.5 mg/kg, and the administration volume was 2 ml/kg.
  • Another group of dogs were given eribulin intravenously, with the dose of 0.5 mg/kg and the administration volume of 2 ml/kg.
  • Dogs were injected with compound 1, and 1 ml of blood was collected before administration and at 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours after administration.
  • the collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice and centrifuged within 1 hour to separate plasma (centrifugal force 2200g, centrifugation 10min, 2-8°C).
  • the plasma samples were stored in a -80°C refrigerator before testing.
  • Dogs were injected with eribulin compounds, and 1 ml of blood was collected before administration and at 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours after administration.
  • the collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice and centrifuged to separate plasma within 1 hour (centrifugal force 2200g, centrifugation 10min, 2-8°C). Plasma samples were stored in a -80°C refrigerator before testing.
  • Test Example 4 Efficacy of ADC-001 on subcutaneous transplanted tumors of human gastric cancer NCI-N87/16-8 nude mice
  • ADC-001 was diluted with saline to the desired concentration.
  • NCI-N87 cells Human gastric cancer NCI-N87 cells were purchased from American Type Culture Collection. NCI-N87 cells were resistant to T-DM1 after long-term induction culture with T-DM1 and named NCI-N87/16-8. The cells were cultured in 10-cm culture dishes in RPMI 1640 medium (Gibco) with 10% fetal bovine serum and penicillin and streptomycin at 37°C in an incubator containing 5% CO2 air. The cells were passaged 2-3 times a week. When the cells were in the exponential growth phase, they were digested with trypsin, collected, counted, and inoculated.
  • BALB/cJGpt-Foxn1 nu /Gpt mice 4 weeks old, female, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Production license number: SCXK (Su) 2019-0009; Animal qualification certificate number: 202004958. Breeding environment: SPF grade.
  • Each nude mouse was subcutaneously inoculated with 1 ⁇ 10 7 NCI-N87/16-8 cells. After the tumor grew to 100-150 mm 3 , the animals were grouped according to tumor volume and body weight (D0). The mice were intravenously injected with 0.3 and 0.6 mg/ml ADC-001, and the administration volume was 10 mL/kg. The tumor volume was measured twice a week, the mouse body weight was weighed, and the data were recorded.
  • the experimental index is to examine the effect of drugs on tumor growth, and the specific index is T/C% or tumor growth inhibition rate TGI (%).
  • Tumor diameter was measured with a vernier caliper twice a week, and tumor volume (V) was calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width respectively.
  • T/C (%) (TT 0 )/ (CC 0 ) ⁇ 100, wherein T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • the growth inhibition rate TGI (%) 100-(TT 0 )/T 0 ⁇ 100
  • T ⁇ T 0 or C ⁇ C 0 it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).
  • the tumor volumes between the two groups were compared using the two-tailed Student’s t test, and P ⁇ 0.05 was defined as a statistically significant difference.
  • ADC-001 at 3 mg/kg and 6 mg/kg could inhibit the growth of NCI-N87/16-8 cells in the nude mouse subcutaneous transplant tumor model, with tumor inhibition rates of 46.77% and 90.18%, respectively (P ⁇ 0.01 vs PBS control).
  • the tumor-bearing mice tolerated ADC-001 well, without any symptoms such as weight loss.
  • ADC-001 can effectively inhibit the growth of human gastric cancer NCI-N87/16-8 cells in the subcutaneous transplanted tumor model of nude mice, and tumor-bearing mice can tolerate ADC-001 well.
  • Test Example 5 Efficacy of ADC-001 on JIMIT-1 cell subcutaneous transplanted tumors in nude mice
  • ADC-001 was diluted with PBS to the desired concentration.
  • mice were subcutaneously inoculated with 5 ⁇ 10 5 JIMT1 cells on the right back. When the tumor volume reached 80-100 mm 3 , they were randomly divided into groups according to the tumor volume and mouse weight, with 6 mice in each group. The drug was administered on the day of grouping. HRA00092-C063-004 was diluted to 0.6 mg/ml with PBS and intravenously injected at a volume of 10 ml/kg. The control group was intravenously injected with the same volume of PBS.
  • the long and short diameters of the tumors were measured with a vernier caliper twice a week, and the body weight of the mice was measured.
  • the tumor volume (V) was calculated as follows:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width respectively.
  • T/C (%) (TT 0 )/ (CC 0 ) ⁇ 100, wherein T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • the tumor volumes between the two groups were compared using the two-tailed Student’s T test, and P ⁇ 0.05 was defined as a statistically significant difference.
  • ADC-001 could significantly inhibit the growth of JIMIT-1 cells in the nude mouse subcutaneous transplant tumor model, with a tumor inhibition rate of 121.3%, and tumors in 5 mice regressed ( ⁇ 50mm 3 ). Tumor-bearing mice tolerated ADC-001 well, without symptoms such as weight loss.
  • 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 stability placement - SEC% of the preparation after stability placement.
  • a method of electrophoresis in which the gel is moved into a capillary tube as a supporting medium and the samples are separated according to their molecular weight at a certain voltage.
  • R-CE% A main peak/A total ⁇ 100% (A main peak is the peak area of the light chain main peak + the heavy chain main peak in the sample, and A total is the sum of all peak areas.)
  • ⁇ R-CE% R-CE% of the preparation before stability placement - R-CE% of the preparation after stability placement.
  • CE measurement instrument Beckman model plus800
  • the freezing point method is used to measure osmotic pressure. It is based on the fact that the freezing point depression value is proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and convert the electricity into osmotic pressure. Instrument manufacturer: Loser, model OM815.
  • the protein used in the following examples is anti-Her2-ADC.
  • Instrument for determining protein concentration UV-visible spectrophotometer, model: Nano Drop 2000, optical path length: 1 mm.
  • the concentration of the antibody drug conjugate in the present disclosure is based on the concentration of the protein, that is, the concentration of the antibody portion in the antibody drug conjugate.
  • a 280nm is the average absorbance of a single sample of the test solution at a wavelength of 280nm when the optical path is 1cm;
  • E mAb-280 The mass extinction coefficient of the protein at a wavelength of 280 nm is 1.45 g -1 cm -1 L;
  • C mAb protein concentration, mg/mL
  • the protein in the sample was removed by ACN precipitation, the supernatant was dried with nitrogen and then re-dissolved.
  • the toxins are separated according to their different polarities, and the toxin content in the sample is calculated using the linear equation formula of toxin standard concentration-peak area fitting.
  • Measuring instrument waters ACQuity H class
  • toxins and related impurities in ADC products were quantitatively determined by liquid chromatography-mass spectrometry. This method directly diluted the sample and loaded it on UPLC-MS for detection. At the same time, the free toxin content in the sample was determined by the external standard curve method combined with the extracted ion flow chromatographic peak area of the toxin standard.
  • a formulation containing the buffer system shown in Table 4, 20 mg/mL protein, 0.2 mg/mL polysorbate 80 (PS80), 40 mg/mL sucrose, and 9 mg/mL glycine was prepared.
  • a forced degradation study (40°C for one month) was performed on the samples, and the effects of different buffer systems on protein stability were investigated using SEC as the evaluation index.
  • His-HCl stands for histidine-histidine hydrochloride
  • CA citric acid-sodium citrate
  • SA succinic acid-sodium succinate
  • 40°CM1 40°C for one month
  • ⁇ SEC% the difference between D0 and 40°CM1SEC monomers, the same below.
  • Preparations containing the buffer system shown in Table 5, 20 mg/mL protein, 0.2 mg/mL PS80, 40 mg/mL sucrose, and 9 mg/mL glycine were prepared.
  • the samples were subjected to a stability study at 2-8°C, and the effects of different pH buffer systems on free toxins were investigated using free toxins as the evaluation index.
  • 2-8°CM1 means storage at 2 ⁇ 8°C for one month.
  • Preparations containing the buffer system shown in Table 6, 20 mg/mL protein, 0.2 mg/mL polysorbate 80 (PS80), 40 mg/mL sucrose, and 9 mg/mL glycine were prepared.
  • the samples were subjected to stability studies (one day at room temperature, one day at 2-8°C, and two cycles of repeated freeze-thaw between -35°C and 2-8°C), and the effects of different pH and buffer systems on free toxins were investigated using free toxins as evaluation indicators.
  • the results are shown in Table 6.
  • the free toxin data showed that as the pH decreased, the growth rate of free toxin slowed down, and the buffer system SA was better than His-AA. Therefore, the buffer system was preferably 30 mM SA pH 4.2.
  • RT24H room temperature for one day
  • 2-8°CD1 means 2-8°C for one day
  • FT2C means two cycles of freezing and thawing between -35°C and 2-8°C
  • ⁇ Free toxin means the increase of free toxin after storage compared with the value at 0, the same below.
  • a solution and a lyophilized powder preparation containing the buffer system shown in Table 7, 20 mg/mL protein, 0.6 mg/ml poloxamer 188 (F68), 40 mg/mL sucrose, and 9 mg/mL glycine were prepared.
  • the samples were subjected to stability studies (solution samples were placed at room temperature for 24 hours, 2-8°C for 3 days, and repeated freeze-thaw cycles between -35°C and 2-8°C for 5 cycles, and lyophilized powder samples were placed at 40°C for 4 weeks).
  • the effects of different buffer system pH on protein stability and free toxins were investigated using appearance, SEC, R-CE, and free toxins as evaluation indicators.
  • the results are shown in Tables 7 and 8.
  • the results show that the stability of the solution: SEC/CE/free toxin was good under pH 4.0-4.2 conditions, and the free toxin at pH 4.6 increased slightly at RT24h and 2-8°CD3, but the overall free toxin was still low, and the stability was acceptable.
  • the lyophilized agent pH 4.0-4.6 had good stability at 4 degrees and 40 degrees for 4 weeks, with no difference between the groups.
  • the buffer system is preferably 30mM SA pH 4.2.
  • D0 means 0 day
  • RT24H means room temperature for 24 hours
  • D3 means 3 days
  • FT1C means one cycle of repeated freezing and thawing between -35°C and 2 ⁇ 8°C, and so on.
  • FR means that the lyophilized powder is reconstituted with water for injection; D0 means 0 day; W4 means 4 weeks.
  • the stability of the samples was studied, and the effects of different concentrations of excipients and osmotic pressure regulators on protein stability were investigated using appearance, SEC, and free toxins as evaluation indicators.
  • RT24H means room temperature for 24 hours
  • 2-8°CD1 means 2-8°C for 1 day
  • F/T1C means one cycle of repeated freezing and thawing between -35°C and 2-8°C
  • N/A means not tested.
  • a solution containing the surfactants shown in Table 11, 30 mM SA pH 4.2, 20 mg/mL protein, 40 mg/mL sucrose, and 9 mg/mL glycine was prepared.
  • the samples were placed in a stable state (7 days at room temperature and 7 days at 4°C), and the effects of different surfactants on protein stability were investigated using SEC and free toxins as evaluation indicators.
  • Solutions and lyophilized powder preparations containing different concentrations of F68, 30 mM SA pH 4.2, 20 mg/mL protein, 40 mg/mL sucrose, and 9 mg/mL glycine as shown in Table 12 were prepared.
  • the stability of the samples was studied (the solution samples were placed at 2-8°C for 3 days, repeatedly frozen and thawed between -35°C and 2-8°C for 3 times, and the product was freeze-dried at 0 hours).
  • the appearance and SEC were used as evaluation indicators to investigate the effect of different concentrations of F68 on protein stability.
  • the surfactant concentration is preferably 0.4-0.6 mg/mL. More preferably, 0.6 mg/ml.

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Abstract

本公开涉及一种含艾日布林衍生物药物偶联物的药物组合物。具体而言，本公开涉及一种药物组合物，包含抗体药物偶联物和缓冲剂，其中所述抗体药物偶联物具有如式I所示的结构，其中Ab为Pertuzumab。本公开的药物组合物具备良好的稳定性。

Description

一种含艾日布林衍生物药物偶联物的药物组合物 技术领域
本公开属于药物制剂领域，具体涉及一种含艾日布林衍生物药物偶联物的药物组合物。
背景技术
抗体药物偶联物(antibody drug conjugate，ADC)把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的药物相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和药物的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响。
微管为与包括细胞内迁移和转运、细胞信号传导和维持细胞形状的多种细胞功能相关的有力的细丝状细胞骨架蛋白。微管也在有丝分裂细胞分裂中通过形成染色体分成两个子细胞所需的有丝分裂纺锤体而起到关键作用。所有细胞中微管的生物功能大部分由其聚合动力学调节，这通过α和β微管蛋白二聚物可逆、非共价地加在微管两端进行。这种动力学行为和所产生的对微管长度的控制为有丝分裂纺锤体的适当功能所不可缺少的。甚至微管动力学的微小改变也会牵涉轴检查点，抑制有丝分裂时细胞周期进展，且随后引起细胞死亡。由于癌细胞的细胞分裂快速，所以与正常细胞相比，其一般对结合于微管蛋白且破坏其正常功能的化合物更加敏感。因此，微管蛋白抑制剂及其抗体药物偶联物有望成为一类有前景的治疗癌症的药物。
ADC具有比抗体更复杂的异质结构，因此，对用于治疗目的ADC制剂提出了更大的挑战。
发明内容
本公开提供一种药物组合物，包含抗体药物偶联物和缓冲剂，其中所述抗体药物偶联物具有如式I所示的结构：
其中：
Ab为Pertuzumab；
n为1至10；
所述缓冲剂选自枸橼酸盐缓冲剂、组氨酸缓冲剂和琥珀酸盐缓冲剂。
在一些实施方案中，所述缓冲剂选自组氨酸-盐酸组氨酸、枸橼酸-枸橼酸钠和琥珀酸-琥珀酸钠。
在一些实施方案中，药物载量(n)的范围可以是每个Pertuzumab抗体结合细胞毒性药物的平均数，非限制性的实施例包括每个抗体结合细胞毒性药物的平均个数为1、2、3、4、5、6、7、8、9、10、11、12以及这些点值之间的任意范围。例如可以是2-8，2-7，2-6，2-5，2-4，3-4，3-5，3.5～4.7，5-6，5-7，5-8和6-8。示例性的，药物载量(n)可以为1，2，3，4，5，6，7，8，9，10的均值。n是小数或整数。在一些实施方案中，n为1至8，或3至5。
在一些实施方案中，所述药物组合物的pH为3.5至5.5，非限制性的实施例包括3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5，以及这些点值之间的任意范围。在一些实施方案中，pH为3.5至5.0，或pH为3.7至4.7。
在一些实施方案中，药物组合物中缓冲剂浓度为5mM至50mM，非限制性的实施例包括5mM、10mM、12mM、15mM、16mM、17mM、18mM、19mM、20mM、30mM、40mM、50mM以及这些点值之间的任意范围；在一些实施方案中，缓冲及浓度为10mM至50mM；在一些实施方案中，缓冲及浓度为20mM至40mM；在一些实施方案中，缓冲及浓度为30mM。
在一些实施方案中，药物组合物还包含表面活性剂。可选自聚山梨酯、泊洛沙姆、聚羟亚烃、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、聚丙二醇和乙烯与丙烯二醇的共聚物等等。在一些实施方案中，表面活性剂是泊洛沙姆或聚山梨酯，例如泊洛沙姆188、聚山梨酯20、聚山梨酯80。
在一些实施方案中，药物组合物中表面活性剂的浓度为0.01mg/mL至1.0mg/mL，或0.1mg/mL至0.8mg/mL，或0.3mg/mL至0.8mg/mL，在一些实施方案中，表面活性剂的浓度为0.6mg/mL，非限制性的实施例包括0.02mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.25mg/mL、0.3mg/mL、0.35mg/mL、 0.4mg/mL、0.45mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL，以及这些点值之间的任意范围。
在一些实施方案中，前述药物组合物还包含糖。本公开的“糖”包含常规组合物(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。所述的糖可选自葡萄糖，蔗糖，海藻糖，α,α-海藻糖二水合物，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，密里二糖，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-麦芽酮糖等等。在一些实施方案中，糖为蔗糖。
在一些实施方案中，前述药物组合物中糖的浓度为25mg/mL至80mg/mL，或者30mg/mL至50mg/mL，非限制性的实施例包括25mg/mL、30mg/mL、35mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、70mg/mL、80mg/mL以及这些点值之间的任意范围。在一些实施方案中，浓度为40mg/mL。
在一些实施方案中，前述药物组合物还包含氨基酸，例如甘氨酸。
在一些实施方案中，前述药物组合物中所述氨基酸的浓度为6mg/mL至15mg/mL，例如，7mg/mL至11mg/mL，或7mg/mL至10mg/mL，非限制性的实施例包括6mg/mL、6.5mg/mL、7mg/mL、7.2mg/mL、7.6mg/mL、7.8mg/mL、8mg/mL、8.5mg/mL、9mg/mL、10mg/mL、10.2mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL以及这些点值之间的任意范围。在一些实施方案中，浓度为9mg/mL。
在一些实施方案中，药物组合物中所述抗体药物偶联物浓度为以蛋白浓度计，1mg/mL至100mg/mL，非限制性的实施例包括1mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL以及这些点值之间的任意范围；在一些实施方案中，所述抗体药物偶联物浓度为以蛋白浓度计，10mg/mL至30mg/mL，例如20mg/mL。具体的，非限制性的实施例包括20.1mg/mL、20.2mg/mL、20.3mg/mL、20.4mg/mL、20.5mg/mL、20.6mg/mL、20.7mg/mL、20.8mg/mL、20.81mg/mL、20.82mg/mL、20.83mg/mL、20.84mg/mL、20.85mg/mL、20.86mg/mL、20.87mg/mL、20.88mg/mL、20.89mg/mL、20.9mg/mL、20.91mg/mL、20.92mg/mL、20.93mg/mL、20.94mg/mL、20.95mg/mL、20.96mg/mL、20.97mg/mL、20.98mg/mL、20.99mg/mL、21mg/mL以及这些点值之间的任意范围。所述的以蛋白浓度计，是指以抗体药物偶联物中的抗体部分的浓度计。
在一些实施方案中，所述的药物组合物，其包含：
(a)以蛋白浓度计，1mg/mL至100mg/mL的所述抗体药物偶联物，(b)0.1 mg/mL至0.8mg/mL的泊洛沙姆或聚山梨酯，(c)30mg/mL至50mg/mL的蔗糖，(d)6mg/mL至15mg/mL甘氨酸，和(e)10mM至50mM的琥珀酸-琥珀酸钠缓冲剂；所述药物组合物的pH为3.5至5.5。
在一些实施方案中，所述的药物组合物，其包含：
(a)以蛋白浓度计，1mg/mL至50mg/mL的所述抗体药物偶联物，(b)0.2mg/mL至0.8mg/mL的泊洛沙姆或聚山梨酯，(c)30mg/mL至50mg/mL的蔗糖，(d)6mg/mL至15mg/mL甘氨酸，和(e)20mM至40mM的琥珀酸-琥珀酸钠缓冲剂；所述药物组合物的pH为3.7至4.7。
在一些实施方案中，所述的药物组合物，其包含：
(a)以蛋白浓度计，20mg/mL的所述抗体药物偶联物，(b)0.6mg/mL的泊洛沙姆188或0.2mg/mL的聚山梨酯80，(c)40mg/mL的蔗糖，(d)9mg/mL甘氨酸和(e)30mM琥珀酸-琥珀酸钠缓冲剂，所述药物组合物的pH为4.2。
在一些实施方案中，前述任一项的药物组合物是液体制剂。本公开的液体制剂或者复溶后的制剂，具有较好的稳定性。进一步的，稳定的液体制剂包括这样的液体制剂：其在包括40℃的温度保存1个月后表现出期望的特征。
本公开还提供一种含抗体药物偶联物的冻干制剂，其中所述制剂复溶后可形成如上所述的药物组合物。
本公开还提供一种制备含抗体药物偶联物的冻干制剂的方法，其中包括将如上所述的药物组合物经冷冻干燥的步骤。
在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天，至少28天或至少30天。
本公开还提供一种包含抗体药物偶联物的冻干制剂，所述冻干制剂通过将如上所述的抗体药物偶联物的药物组合物冷冻干燥获得。
本公开还提供一种含抗体药物偶联物的复溶溶液，其中所述复溶溶液是通过将如上所述的冻干制剂复溶制备获得。
本公开还提供制备上述复溶溶液的方法，其中包括将前述冻干制剂经复溶的步骤，其复溶所用溶液选自但不限于注射用水、生理盐水或葡萄糖溶液。
本公开还提供一种制品，其包括容器，该容器中装有如上所述的药物组合物、冻干制剂或复溶溶液。在一些实施方案中，该容器为中性硼硅玻璃管制注射剂瓶。
本公开还提供前述的药物组合物或冻干制剂或复溶溶液或制品在制备治疗或预防肿瘤的药物中的应用。
本公开还提供治疗疾病的方法，包括向有需要的患者施用前述的药物组合物或冻干制剂或复溶溶液或制品。
本公开还提供作为药物的前述的药物组合物，或冻干制剂，或复溶溶液，或制品，用于治疗疾病。
本公开还提供作为药物的前述的药物组合物，或冻干制剂，或复溶溶液，或 制品，用于治疗肿瘤。
本公开还提供作为药物的前述的药物组合物，或冻干制剂，或复溶溶液，或制品，在制备治疗肿瘤的药物中的用途。
在一些实施方案中，其中所述的肿瘤为与HER2的结构域II表达相关的癌症。
在一些实施方案中，其中所述肿瘤选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌和淋巴瘤。
以下为Pertuzumab的重、轻链的可变区序列：
轻链可变区
重链可变区
以下为Pertuzumab的序列：
轻链
重链
如本领域技术人员所熟知的，本公开中所述各个实施方案的一项、一些或所有特性可以进一步组合以形成本公开的其它实施方案。本公开的以上实施方案和通过组合得到的其他实施方案通过下面的详述进一步说明。
发明详述
本公开提供一种更利于生产和给药，性能稳定的药物组合物。其中，不期望的不稳定性可包括下列任何一项或多项：聚集、脱酰胺化(例如Asn脱酰胺化)、氧化(例如Met氧化)、异构化(例如Asp异构化)、剪裁(clipping)/水解/片段化(例如铰链区片段化)、琥珀酰亚胺形成、不成对的半胱氨酸、毒素的解离等。具体地，本公开所述的药物组合物包含抗体药物偶联物和缓冲剂。
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
“抗体药物偶联物(antibody drug conjugate，ADC)是把抗体通过连接单元与具有生物活性的细胞毒素或具有细胞杀伤活性的小分子药物相连。
“载药量”或“药物载量”也称药物抗体比例(Drug-to-Antibody Ratio，DAR)，即ADC中每个抗体所偶联的药物的平均数量。其可在例如每个抗体偶联1至10个药物的范围内，并且在某些实施例中，在每个抗体偶联1至8个药物的范围内，优选自2-8，2-7，2-6，2-5，2-4，3-4，3-5，5-6，5-7，5-8和6-8。示例性的，载药量可以为1，2，3，4，5，6，7，8，9，10的均值。本公开的ADC通式包括与前述一定范围内的药物偶联的抗体的集合。在本公开的实施方式中，载药量可表示为n。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC测定载药量。
术语“接头单元”或“连接片段”或“连接单元”是指一端与抗体或其抗原结合片段连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域已知方法合成，诸如US20050238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“药物连接臂片段”或“药物-接头片段”是指药物与接头单元相连形成的片 段，其可通过接头单元另一端与抗体相连。
可以用以下非限制性方法控制细胞毒性药物的载量，包括：
(1)控制连接药物连接臂片段和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
本公开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。
本公开所述的“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。通常，天然的完整抗体由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“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-琥珀酸钠可由琥铂酸与氢氧化钠配制而成，或由琥铂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磷酸氢二钠-磷酸二氢钠。
“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。
“药物组合物”表示含有一种或多种本文所述抗体药物偶联物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持抗体活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本公开中，“药物组合物”和“制剂”并不互相排斥。
本公开中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“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。
本文所用术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开中数值为仪器测量值或仪器测量后计算值，存在一定程度的误差，一般而言，正负10％均属于合理误差范围内。当然需要考虑该数值所用之处的上下文，例如，总杂质的含量，该数值为测量后误差变化不超过正负10％，可以为正负9％、正负8％、正负7％、正负6％、正负5％、正负4％、正负3％、正负2％或正负1％，优选正负5％。
本公开所述的药物组合物能够达到一种稳定的效果：其中的抗体药物偶联物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或抗体药物偶联物稳定性的分析技术，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3个月、优选6个月、更优选1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、2个月、3个月在内的时段后表现出期望的特征。进一步的，稳定的液体制剂包括这样的液体制剂：其在包括40℃ 的温度保存包括10天、20天、1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％的抗体单体发生聚集或降解。通过视觉分析，制剂是淡黄色近无色澄明液体或者无色，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的减少。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，抗体药物偶联物没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体药物偶联物在药物制剂中“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。
如果抗体药物偶联物没有显示出显著的化学改变，那么所述抗体药物偶联物在药物制剂中“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)。
如果抗体药物偶联物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，那么所述抗体药物偶联物在药物制剂中“保留它的生物活性”。
“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。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
常规的药物组合物的制备见中国药典。
术语“载体”用于本公开的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
“给予”和“处理”当应用于动物、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。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“置换”是指溶解抗体蛋白或抗体药物偶联物的溶剂体系的置换，例如，使用稳定制剂的缓冲体系经物理操作方式将含抗体蛋白或抗体药物偶联物的高盐或高渗溶剂体系置换，从而使抗体蛋白或抗体药物偶联物存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
在本文中，“Pertuzumab”指包含分别在SEQ ID No.1和2中的轻链和重链可变区氨基酸序列的抗体。若Pertuzumab是完整抗体，则它优选包含分别在SEQ ID No.3和4中的轻链和重链氨基酸序列。
附图说明
图1为N87/16-8皮下移植瘤模型小鼠肿瘤体积变化图。
图2为N87/16-8皮下移植瘤模型小鼠体重变化图。
图3为JIMIT-1皮下移植瘤模型小鼠肿瘤体积变化图。
图4为JIMIT-1皮下移植瘤模型小鼠体重变化图。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非是对本公开范围的限制。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如参照冷泉港实验室出版的《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。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl3)、氘代甲醇(CD3OD)，内标为四甲基硅烷(TMS)。
MS的测定用Agilent 1200/1290 DAD-6110/6120 Quadrupole MS液质联用仪(生产商：Agilent，MS型号：6110/6120 Quadrupole MS)。
waters ACQuity UPLC-QD/SQD(生产商：waters，MS型号：waters ACQuity Qda Detector/waters SQ Detector)THERMO Ultimate 3000-Q Exactive(生产商：THERMO，MS型号：THERMO Q Exactive)
高效液相色谱法(HPLC)分析使用Agilent HPLC 1200DAD、Agilent HPLC 1200VWD和Waters HPLC e2695-2489高压液相色谱仪。
手性HPLC分析测定使用Agilent 1260 DAD高效液相色谱仪。
高效液相制备使用Waters 2545-2767、Waters 2767-SQ Detecor2、Shimadzu LC-20AP和Gilson GX-281制备型色谱仪。
手性制备使用Shimadzu LC-20AP制备型色谱仪。
CombiFlash快速制备仪使用Combiflash Rf200(TELEDYNE ISCO)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm至0.2mm，薄层层析分离纯化产品采用的规格是0.4mm至0.5mm。
硅胶柱色谱法一般使用烟台黄海硅胶200至300目硅胶为载体。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃至30℃。
纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂的体系包括：A：二氯甲烷和异丙醇体系，B：二氯甲烷和甲醇体系，C：石油醚和乙酸乙酯体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和酸性或碱性试剂等进行调节。
本文所述的如式I所示的抗体药物偶联物可依照PCT/CN2022/107479的方法制备。
一、抗体偶联物的制备
实施例1
步骤1：化合物1b的制备
冰水浴下，将化合物1a(艾日布林，参照ZL201010236637.2方法制备获得)(72.91mg，0.1mmol)溶于10mL的四氢呋喃中，加入Fmoc-OSu(芴甲氧羰酰琥珀酰亚胺，41mg，0.12mmol)，然后在室温搅拌至反应完全。减压浓缩得粗品，直接用于下一步反应。
步骤2：化合物1c的制备
将上步所得化合物1b的粗品溶于10mL无水乙醚中，加入氧化银(34.8mg，0.15mmol)，接着加入碘甲烷(28.4mg,0.2mmol)，然后于室温搅拌至反应完全，过滤，然后减压浓缩得粗品，直接进行下一步反应。
步骤3：化合物1的制备
将上步所得化合物1c粗品溶于10mL的四氢呋喃中，加入2mL的二乙胺，然后在室温搅拌至反应完全，减压浓缩得粗品，经硅胶柱层析色谱法(洗脱剂：二氯甲烷/乙酸乙酯/石油醚)纯化，得3mg目标产物化合物1。
LC/MS(ESI)：m/z 744.2[M+H]+。
冰水浴下，将4a(50mg,0.08mmol，参照WO2017151979中方法制备)溶解到1.5mL的N,N-二甲基甲酰胺中，然后加入DIPEA(N,N-二异丙基乙胺，18mg,0.14mmol)，接着加入二(对硝基苯)碳酸酯(49mg,0.16mmol)，然后在室温下搅拌10加入20mL甲基叔丁基醚，搅拌20分钟，过滤，干燥得到固体36mg，LC/MS(ESI):m/z 784.1[M+H]+。
将化合物1(13.5mg,0.018mmol)溶于1.5mL的DMF中，加入DIPEA(7mg,0.054mmol)，然后分批加入化合物4b(18mg,1.3mmol)，搅拌至反应基本完全，浓缩得粗品，经HPLC制备分离得6.5mg化合物L-1，纯度96.95％，LC/MS(ESI):m/z 1388.3[M+H]+。
实施例2 ADC-001
在37℃条件下，向抗体Pertuzumab的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.43mL，97nmol)加入配置好的的三(2-羧乙基)膦盐酸盐(TCEP-HCl)的水溶液(10mM，24.2uL，242nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物L-1(1.34mg，965nmol)溶解于60uL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH＝6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物的PBS缓冲液(1.24mg/mL，9.8mL)，于4℃冷藏储存。
CE-SDS计算平均值：n＝3.17。
实施例3 ADC-001
在12℃条件下，在含有2.5mM EDTA的20mM组氨酸-盐酸-Tris缓冲液(pH 7.2，7.73L)中，含135.32g Pertuzumab抗体(0.93mmol)与0.5880g三(2-羧乙基)膦盐酸盐(Sigma，2.05mmol)在恒温水浴中搅拌反应120min，生成中间体I溶液。滴加2M醋酸调节反应液pH至5.2。
将化合物L-1(6.865g，4.94mmol)溶解于0.425L DMSO中，生成化合物L-1的DMSO溶液。向上述中间体I溶液中预加0.348L DMSO，再将上述化合物L-1的DMSO溶液加入到预加DMSO的中间体I溶液中，于水浴12℃下搅拌。滴加2M醋酸调节反应液pH至4.2。
将上述反应液经0.22μm滤器过滤后，使用琥珀酸缓冲液(pH＝4.2)超滤，得到产物ADC-001(20g/L，反相色谱计算平均值：n＝4.1)。
二、生物学测试
测试例1：对体外培养人乳腺癌BT-474和人胃癌NCI-N87,NCI-N87/16-8,NCI-N87/8-2细胞增殖的抑制作用
1.1药物信息
注：以上样品由上海恒瑞有限公司提供。其中帕妥珠单抗按照WO2006033700中类似方法制备获得；kadcyla：曲妥珠单抗-美坦新偶联物。
1.2受试细胞
NCI-N87和BT-474细胞购自American Type Culture Collection(ATCC)。T-DM1长期作用NCI-N87诱导出的T-DM1耐药的NCI-N87/16-8和NCI-N87/8-2细胞由本实验室构建。细胞用含10％胎牛血清(FBS)的RPMI 1640/DMEM(1：1)培养液培养。
1.3仪器及试剂
RPMI 1640和DMEM购自Gibco BRL公司；FBS购自Gibco公司；磺酰罗丹明B(SRB)购自Sigma公司。
酶标仪Synergy H4购自BioTek公司。
1.4实验步骤
接种一定数量的对数生长期细胞于96孔培养板。贴壁生长24小时后，加入不同浓度(10000,3000,1000,300,100,30,10,3,1ng/mL)的药物。药物处理120小时后，用三氯乙酸固定细胞。然后SRB溶液染色；最后加入Tris溶液溶解SRB，酶标仪510nm波长下测定OD值，以下列公式计算细胞生长抑制率：
抑制率＝(OD值对照孔－OD值给药孔)/OD值对照孔×100％
根据各浓度抑制率，用GraphPad Prism 7软件计算半数抑制浓度IC50。
表1.对HER2阳性肿瘤细胞增殖抑制作用IC50(ng/mL)
测试例2：化合物1的体外细胞毒活性筛选
1.1.实验原理及方法
本实验利用CTG检测ATP含量，反映肿瘤细胞的存活情况。首先通过种植不同密度的细胞并培养3天和5天，根据IC50和最大抑制率确定最终培养条件。然后按照此条件检测毒素分子的杀伤作用。
1.2.细胞株的选择
根据实验目的，选择乳腺癌、NSCLC两种疾病模型，选出SKBR3肿瘤细胞(HER2+，ATCC，货号HTB-30)、MDA-MB-468(HER2-，ATCC，货号HTB-132)和A549(人非小细胞肺癌细胞，ATCC，货号CCL-185)三株细胞用于筛选实验。
1.3.细胞培养条件的确定
1)细胞培养：A549、SK-BR-3和MDA-MB-468细胞分别用含10％FBS(Gibco，10099-141)的Ham’s F-12K(Kaighn’s)培养基(Gibco，21127030)和McCoy's 5A培养 基(ThermoFisher，货号16600108)培养及Leibovitz's L-15培养基(ThermoFisher，货号11415-114)进行培养。
2)细胞铺板：将A549用胰酶消化后，用上述培养基终止，计数后分别取4.3×105、7.2×105、11.5×105个细胞加培养液使终体积均为26ml。在96孔板(康宁，货号3903)第2列到第11列每孔加180ul细胞悬液，使得细胞密度分别为每孔3K、5K、8K。第12列为200ul培养液，剩余孔用PBS填充。SKBR3和MDA-MB-468细胞重复上述操作。平行两份。
2)药物配制：在圆底96孔板(康宁，货号3788)中，用DMSO配制阳性对照艾日布林和本公开化合物储备液。在配药板1的第1列配制2mM(用DMSO将储液稀释10倍)，此后到第10列梯度10倍稀释于DMSO中，第11列为DMSO。配药板2第2列至第11列每孔加入相应培养液95ul，从配药板1的第2列至第11列吸取5ul溶液至配药板2，混匀后，吸取20ul加入铺好的细胞中，继续培养3天和5天。
3)CTG检测(Cell Titer-GloTM，发光细胞生存能力检测，Promega公司)：分别在第3天和第5天取出细胞板，平衡至室温。每孔加入90ul CTG，避光室温反应10min，酶标仪读取发光值并计算IC50。
1.4.数据结果
表2
测试例3：化合物1药代动力学测试
1、概述
以的比格犬为受试动物，应用LC/MS/MS法测定了比格犬静脉注射给予化合物1和艾日布林后不同时刻血浆中的药物浓度。研究本公开化合物在犬体内的药代动力学行为，评价其药动学特征。
2、试验方案
2.1试验药品
化合物1和艾日布林
2.2试验动物
比格犬6只，雄性，平均分为2组，由美迪西普亚医药科技(上海)有限公司采购并进行动物给药实验。
2.3药物配制
称取化合物1，加5％体积的DMSO，20％PG和20％PEG400使其溶解，然后加入55％生理盐水配制成0.25mg/ml无色澄明溶液。
称取艾日布林，加5％体积的DMSO，20％PG和20％PEG400使其溶解，然后加入55％生理盐水配制成0.25mg/ml无色澄明溶液。
2.4给药
一组犬静脉注射给药化合物1，给药剂量均为0.5mg/kg，给药体积均为2ml/kg。
另一组犬静脉注射给药艾日布林，给药剂量均为0.5mg/kg，给药体积均为2ml/kg。
3、操作
犬注射给药化合物1，于给药前及给药后5分钟、0.25、0.5、1.0、2.0、4.0、8.0、12.0、24.0小时采血1ml，将收集的血液样本置于EDTA-K2抗凝型采血管中，将收集到的全血放在冰上，1小时内离心分离血浆(离心力2200g，离心10min，2-8℃)。血浆样本在检测前存放于-80℃冰箱内。
犬注射给药艾日布林化合物，于给药前及给药后5分钟、0.25、0.5、1.0、2.0、4.0、8.0、12.0、24.0小时采血1ml，将收集的血液样本置于EDTA-K2抗凝型采血管中，将收集到的全血放在冰上，1小时内离心分离血浆(离心力2200g，离心10min，2-8℃)。血浆样本在检测前存放于-80℃冰箱内。
测定药物注射给药后犬血浆中的待测化合物含量：取给药后各时刻的犬血浆25μl，加入内标溶液喜树碱(中国生物制品检定所)50μl(100ng/mL)和乙腈200μl，涡旋混合5分钟，离心10分钟(3700转/分钟)，血浆样品取上清液3-4μl进行LC/MS/MS(API4000三重四极杆串联质谱仪(No.2)，美国Applied Biosystems公司；Shimadzu LC-30AD超高效液相色谱系统，日本Shimadzu公司)分析。
4、药代动力学参数结果
本公开化合物的药代动力学参数如下表3所示。
表3
测试例4：ADC-001对人胃癌NCI-N87/16-8裸小鼠皮下移植瘤的疗效
1、实验目的
评价ADC-001对人胃癌NCI-N87/16-8裸小鼠皮下移植瘤的疗效。
2、试验药品
ADC-001，用生理盐水稀释至所需浓度。
3、细胞
人胃癌NCI-N87细胞购自American Type Culture Collection。NCI-N87经T-DM1长期诱导培养对T-DM1耐药，命名为NCI-N87/16-8。细胞用10-cm培养皿培养，培养条件为RPMI 1640培养基(Gibco)中加10％胎牛血清以及青、链霉素，于37℃、含5％CO2空气的培养箱中培养。一周2-3次传代，当细胞呈指数生长期时，胰酶消化，收集细胞，计数，接种。
4、试验动物
BALB/cJGpt-Foxn1nu/Gpt小鼠，4周，雌性，购自江苏集萃药康生物科技有限公司。生产许可证号：SCXK(苏)2019-0009；动物合格证号202004958。饲养环境：SPF级。
5、试验步骤
每只裸小鼠皮下接种1×107NCI-N87/16-8细胞，待肿瘤生长至100-150mm3后，根据肿瘤体积和体重将动物分组(D0)。小鼠静脉注射给予0.3和0.6mg/ml的ADC-001，给药体积10mL/kg。每周测2次肿瘤体积，称小鼠体重，记录数据。
6、试验指标
实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或肿瘤生长抑制率TGI(％)。
每周二次用游标卡尺测量肿瘤直径,肿瘤体积(V)计算公式为：
V＝1/2×a×b2其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
生长抑制率TGI(％)＝100-T/C(％)。
当肿瘤出现消退时，生长抑制率TGI(％)＝100-(T-T0)/T0×100
如果肿瘤比起始体积缩小，即T<T0或C<C0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
实验结束、达到实验终点、或溶剂组平均肿瘤体积达到1500mm3，CO2麻醉处死动物，随后解剖取瘤并拍照。
7、统计学分析
除非特别说明，二组肿瘤体积之间比较采用双尾Student’s t检验，P<0.05定义为有统计学显著性差异。
8、结果
单次给药后第22天，3mg/k和6mg/kg，ADC-001可以抑制NCI-N87/16-8细胞在裸小鼠皮下移植瘤模型中的生长，抑瘤率分别为46.77％和90.18％(P<0.01 vs  PBS control)。荷瘤小鼠对能ADC-001很好耐受，没有体重减轻等症状发生。
9、结论
ADC-001能够有效抑制人胃癌NCI-N87/16-8细胞在裸小鼠皮下移植瘤模型中的生长，荷瘤小鼠对ADC-001能很好耐受。
测试例5：ADC-001对JIMIT-1细胞在裸小鼠皮下移植瘤的疗效
1、试验药品
ADC-001，用PBS稀释至所需浓度。
2、细胞
人乳腺癌JIMIT-1细胞。
4、试验动物
雌性Balb/c nude小鼠
5、试验步骤
BALB/c nude小鼠右侧背部皮下接种JIMT1细胞5×105JIMT1细胞，待肿瘤体积达80-100mm3时，根据肿瘤体积和小鼠体重随机分组，每组6只，分组当天开始给药。HRA00092-C063-004采用PBS稀释至0.6mg/ml静脉注射给药，给药体积为10ml/kg，对照组静脉注射给予相同体积的PBS。
每周二次用游标卡尺测量肿瘤长径和短径，并测量小鼠体重。
肿瘤体积(V)计算公式为：
V＝1/2×a×b2其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
生长抑制率TGI(％)＝100-T/C(％)。
除非特别说明，二组肿瘤体积之间比较采用双尾Student’s T检验，P<0.05定义为有统计学显著性差异。
6、结果
单次给药后第24天，ADC-001可以显著抑制JIMIT-1细胞在裸小鼠皮下移植瘤模型中的生长，抑瘤率为121.3％，其中5只小鼠中的肿瘤都出现了消退(<50mm3)。荷瘤小鼠对能ADC-001很好耐受，没有体重减轻等症状发生。
三、制剂
制剂制备与检测过程中使用的设备及结果计算方法如下：
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％＝稳定性放置前制剂的SEC％-稳定性放置后制剂的SEC％。
SEC测定用仪器：安捷伦1260；柱子：waters，XBridgeSEC(300×7.8mm 3.5μm)
R-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。
R-CE％＝A主峰/A总×100％(A主峰为样品中轻链主峰+重链主峰的峰面积，A总为所有峰面积之和。)ΔR-CE％＝稳定性放置前制剂的R-CE％-稳定性放置后制剂的R-CE％。
CE测定用仪器：Beckman型号plus800
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。仪器厂家罗泽Loser，型号OM815。
蛋白浓度：
以下实施例所采用的蛋白为抗Her2-ADC。
蛋白的浓度测定用仪器：紫外可见分光光度计，型号：Nano Drop 2000，光程为1mm。
本披露中的抗体药物偶联物浓度以蛋白的浓度计，即以抗体药物偶联物中抗体部分的浓度。
因为抗体药物偶联物中的毒素在蛋白特征吸收波长280nm下几乎没有吸收，同时该毒素在370nm下几乎没有吸收，故用以下公式对上述蛋白浓度进行计算：
A280nm＝(CmAb×EmAb-280)×l
即：
式中，A280nm：供试品溶液单份样品在光程为1cm时在280nm波长处的吸光度平均值；
EmAb-280：蛋白在280nm波长处的质量消光系数，为1.45g-1cm-1L；
CmAb：蛋白的浓度，mg/mL；
l：光程长度，cm(此处光程为1cm)。
若供试液经过稀释，则蛋白浓度为：C(mg/mL)＝CmAb×N，N为稀释倍数。
游离毒素测定(RP-UPLC反相法):
将样品中的蛋白用ACN沉淀除去，取上清氮气吹干后复溶，根据样品中的毒 素的极性不一样进行分离，再通过毒素标准品浓度-峰面积拟合的线性方程公式计算样品中的毒素含量。
测定仪器：waters ACQuity H class；
色谱柱：waters ACQUITY UPLC BEH Shield RP18，1.7μm，2.1*150mm
游离毒素测定(LC-MS法):
本研究通过液相色谱联用质谱法对ADC产品中的毒素及相关杂质进行定量测定。本方法直接将样品稀释后上样UPLC-MS进行检测，同时通过外标曲线法结合毒素标准品的提取离子流色谱峰面积测定检测样品中的游离毒素含量。
仪器：Waters,Acquity UPLC_Rda(Bioaccord)
柱子：Waters,ACQUITY UPLC BEH C4,1.7μm,2.1×50mm
制剂实施例1.pH和缓冲体系筛选
制备含表4所示的缓冲体系、20mg/mL蛋白、0.2mg/mL聚山梨酯80(PS80)、40mg/mL蔗糖、9mg/mL甘氨酸的制剂。对样品进行强制降解研究(40℃放置一个月)，以SEC为评价指标，考察不同缓冲体系对蛋白稳定性的影响。
结果见表4。SEC数据显示，40℃放置一个月后，含His-HCl或SA的制剂SEC单体下降幅度小于CA组。优选His-HCl和SA体系。
表4 pH和缓冲体系筛选结果
注：His-HCl代表组氨酸-盐酸组氨酸；CA代表枸橼酸-枸橼酸钠盐；SA代表琥珀酸-琥珀酸钠盐；40℃M1：40℃放置一个月，△SEC％：D0与40℃M1SEC单体差值，下同。
制剂实施例2.缓冲体系pH对游离毒素的影响
制备含表5所示的缓冲体系、20mg/mL蛋白、0.2mg/mL PS80、40mg/mL蔗糖、9mg/mL甘氨酸的制剂。对样品进行2～8℃稳定性研究，以游离毒素为评价指标，考察不同pH缓冲体系对游离毒素的影响。
结果见表5。游离毒素数据显示，2～8℃放置一个月后，游离毒素随着缓冲体系pH的降低而减少。pH5.5优于pH6.5.
表5 pH对游离毒素的影响
注：2-8℃M1表示2～8℃放置一个月。
制剂实施例3.pH和缓冲体系对游离毒素的影响
制备含表6所示的缓冲体系、20mg/mL蛋白、0.2mg/mL聚山梨酯80(PS80)、40mg/mL蔗糖、9mg/mL甘氨酸的制剂。对样品进行稳定性研究(室温放置一天、2-8℃放置一天、-35℃与2～8℃之间反复冻融2个循环)，以游离毒素为评价指标，考察不同pH和缓冲体系对游离毒素的影响。
结果见表6。游离毒素数据显示，随着pH的降低，游离毒素的增长速度变慢，缓冲体系SA优于His-AA。因此缓冲体系优选为30mM SA pH4.2。
表6 pH和缓冲体系对游离毒素的影响结果
注：RT24H表示室温放置一天，2-8℃D1表示2-8℃放置一天，FT2C表示-35℃与2～8℃之间反复冻融2个循环，△游离毒素表示放置后游离毒素与0时比增加的值，同下。
制剂实施例4.缓冲体系pH的优化
制备含表7所示的缓冲体系、20mg/mL蛋白、0.6mg/ml泊洛沙姆188(F68)、40mg/mL蔗糖、9mg/mL甘氨酸的溶液剂和冻干粉制剂。对样品进行稳定性研究(溶液剂样品室温放置24小时、2-8℃放置3天、-35℃与2～8℃之间反复冻融5个循环，冻干粉样品40℃放置4周)，以外观、SEC、R-CE、游离毒素为评价指标，考察不同缓冲体系pH对蛋白稳定性和游离毒素的影响。
结果见表7、8。结果显示，溶液剂：pH4.0-4.2各条件下SEC/CE/游离毒素稳定性均较好，pH4.6游离毒素RT24h及2-8℃D3略有增长，但整体游离毒素依然较低，稳定性可以接受。冻干剂pH4.0-4.6 4度及40度放置4周稳定性较好，组间无差异。综合溶液及冻干结果，缓冲体系优选为30mM SA pH4.2。
表7.缓冲体系pH的优化溶液剂结果

注：D0表示0天，RT24H表示室温24小时，D3表示3天，以此类推；FT1C表示-35℃与2～8℃之间反复冻融1个循环，以此类推。
表8缓冲体系pH的优化冻干粉结果
注：FR表示冻干粉用注射用水进行复溶；D0表示0天；W4表示4周。
制剂实施例5.赋形剂及渗透压调节剂的筛选
制备含表9所示赋形剂及渗透压调节剂、30mM SA pH 4.2、20mg/mL蛋白、0.2mg/mL PS80的溶液剂。对样品进行稳定性研究，以外观、SEC和游离毒素为评价指标，考察不同浓度的赋形剂及渗透压调节剂对蛋白稳定性的影响。
结果见表10。RT24H/2-8℃D1/FT1C数据显示，组间外观和SEC无显著性差异。但40mg/mL蔗糖+9mg/mL甘氨酸组游离毒素略优，因此优选40mg/mL蔗糖+9mg/mL甘氨酸。
表9赋形剂及渗透压调节剂信息表
表10赋形剂及渗透压调节剂的筛选结果
注：RT24H表示室温放置24小时；2-8℃D1表示2-8℃放置1天；F/T1C表示-35℃与2～8℃之间反复冻融1个循环；N/A表示未检测。
制剂实施例6.表面活性剂筛选
制备含表11所示表面活性剂、30mM SA pH 4.2、20mg/mL蛋白、40mg/mL蔗糖、9mg/mL甘氨酸的溶液剂。对样品进行稳定行放置(室温放置7天和4℃放置7天)，以SEC和游离毒素为评价指标，考察不同表面活性剂对蛋白稳定性的影响。
结果见表11。数据显示，组间游离毒素、SEC纯度无显著性区别。
表11表面活性剂种类筛选结果
制剂实施例7.表面活性剂浓度筛选
制备含表12所示不同浓度的F68、30mM SA pH 4.2、20mg/mL蛋白、40mg/mL蔗糖、9mg/mL甘氨酸的溶液剂和冻干粉制剂。对样品进行稳定性研究(溶液样品在2-8℃放置3天、-35℃和2～8℃之间反复冻融3次，冻干后产品0时)，以外观和SEC为评价指标，考察不同浓度的F68对蛋白稳定性的影响。
结果见表12。数据显示，对溶液剂，0.4-0.6mg/mL F68处方在不同条件下，样品外观较好，SEC纯度项无显著性差异。冻干复溶后外观和SEC纯度项无显著差异。因此，表面活性剂浓度优选0.4-0.6mg/mL。更优选0.6mg/ml。
表12表面活性剂浓度筛选结果

Claims (16)

  1. 一种药物组合物，包含抗体药物偶联物和缓冲剂，其中所述抗体药物偶联物具有如式I所示的结构：
    其中：
    Ab为Pertuzumab；
    n为1至10，优选为1至8，更优选为3至5；
    所述缓冲剂选自枸橼酸盐缓冲剂、组氨酸缓冲剂和琥珀酸盐缓冲剂，优选组氨酸-盐酸组氨酸、枸橼酸-枸橼酸钠和琥珀酸-琥珀酸钠。
  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3.5至5.5，优选pH为3.5至5.0，更优选pH为3.7至4.7。
  3. 根据权利要求1所述的药物组合物，其中所述缓冲剂的浓度为5mM至50mM，优选为20mM至40mM，更优选为30mM。
  4. 根据权利要求1或2所述的药物组合物，其中所述药物组合物还包含表面活性剂，所述表面活性剂优选为聚山梨酯或泊洛沙姆。
  5. 根据权利要求4所述的药物组合物，其中所述表面活性剂浓度为0.01mg/mL至1.0mg/mL，优选0.05mg/mL至1.0mg/mL，更优选为0.1mg/mL至1.0mg/mL。
  6. 根据权利要求1至5中任一项所述的药物组合物，其中所述药物组合物还包含糖，所述糖优选为蔗糖。
  7. 根据权利要求6所述的药物组合物，其中所述糖浓度为25mg/mL至80mg/mL，优选为30mg/mL至50mg/mL，更优选为40mg/mL。
  8. 根据权利要求1至7中任一项所述的药物组合物，其中所述药物组合物还 包含氨基酸，所述氨基酸优选为甘氨酸。
  9. 根据权利要求8所述的药物组合物，其中所述氨基酸的浓度为6mg/mL至15mg/mL，优选为7mg/mL至11mg/mL，更优选为9mg/mL。
  10. 根据权利要求1至9中任一项所述的药物组合物，其中所述抗体药物偶联物浓度为以蛋白浓度计，1mg/mL至100mg/mL，
    优选地，所述抗体药物偶联物浓度为以蛋白浓度计，10mg/mL至30mg/mL，
    更优选地，所述抗体药物偶联物浓度为以蛋白浓度计，18mg/mL至22mg/mL。
  11. 根据权利要求1至10中任一项所述的药物组合物，其包含：
    (a)以蛋白浓度计，1mg/mL至100mg/mL的所述抗体药物偶联物，(b)0.1mg/mL至0.8mg/mL的泊洛沙姆或聚山梨酯，(c)30mg/mL至50mg/mL的蔗糖，(d)6mg/mL至15mg/mL甘氨酸，和(e)10mM至50mM的琥珀酸-琥珀酸钠缓冲剂；所述药物组合物的pH为3.5至5.5，
    优选的，所述的药物组合物包含：
    (a)以蛋白浓度计，1mg/mL至50mg/mL的所述抗体药物偶联物，(b)0.4mg/mL至0.8mg/mL的泊洛沙姆或聚山梨酯，(c)30mg/mL至50mg/mL的蔗糖，(d)6mg/mL至15mg/mL甘氨酸，和(e)20mM至40mM的琥珀酸-琥珀酸钠缓冲剂；所述药物组合物的pH为3.7至4.7
    更优选的，所述的药物组合物包含：
    (a)以蛋白浓度计，20mg/mL的所述抗体药物偶联物，(b)0.6mg/mL的泊洛沙姆188或0.2mg/mL的聚山梨酯80，(c)40mg/mL的蔗糖，(d)9mg/mL甘氨酸和(e)30mM琥珀酸-琥珀酸钠缓冲剂，所述药物组合物的pH为4.2。
  12. 一种含抗体药物偶联物的冻干制剂，其中所述冻干制剂复溶后可形成权利要求1至11中任一项所述的药物组合物。
  13. 一种含抗体药物偶联物的冻干制剂，其中所述冻干制剂通过将权利要求1至11中任一项所述的药物组合物冷冻干燥获得。
  14. 一种含抗体药物偶联物的复溶溶液，其中所述复溶溶液是通过将权利要求12或13所述的冻干制剂复溶制备获得。
  15. 一种制品，其包括容器，该容器中装有如权利要求1至11中任一项所述的药物组合物、权利要求12或13所述的冻干制剂或权利要求14所述的复溶溶液。
  16. 根据权利要求1至11中任一项所述的药物组合物、根据权利要求12或13所述的冻干制剂、根据权利要求14所述的复溶溶液或权利要求15所述制品在制备治疗病毒感染、肿瘤或癌症的药物中的用途；优选的，所述的肿瘤为与HER2的结构域II表达相关的癌症；优选的，所述肿瘤选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌和淋巴瘤。
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