Pharmaceutical compositions containing eribulin derivative drug conjugates

Abstract

The present disclosure relates to a pharmaceutical composition comprising an eribulin derivative-drug conjugate. Specifically, the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has a structure shown in Formula I, where Ab is Pertuzumab. The pharmaceutical composition of the present disclosure has good stability. JPEG2026504859000023.jpg49170

Description

[Technical Field]

[0001] The present disclosure belongs to the field of drug formulations, and specifically relates to pharmaceutical compositions comprising eribulin derivative drug conjugates. [Background technology]

[0002] Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active drugs via stable chemical linker compounds, taking advantage of the specificity of antibodies in binding to surface antigens on normal cells and tumor cells and the high performance of drugs, while avoiding the drawbacks of the former, such as the relatively low therapeutic efficacy, and the latter, such as excessive toxicity and side effects. This means that, compared with traditional chemotherapy drugs, antibody drug conjugates can bind precisely to tumor cells and reduce their impact on normal cells.

[0003] Microtubules are powerful filamentous cytoskeletal proteins involved in various cellular functions, including intracellular movement and transport, cell signaling, and cell shape maintenance. Microtubules also play a crucial role in mitotic cell division by forming the mitotic spindle, which is required for the division of chromosomes into two daughter cells. Most of the biological functions of microtubules in all cells are regulated by their polymerization dynamics, which are achieved by the reversible, noncovalent binding of α- and β-tubulin dimers to both ends of the microtubule. This dynamic behavior and the resulting control over microtubule length are essential for the proper functioning of the mitotic spindle. Even subtle changes in microtubule dynamics can trigger axial checkpoints, inhibit cell cycle progression during mitosis, and subsequently cause cell death. Because cancer cells divide rapidly, they are generally more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. Therefore, tubulin inhibitors and their antibody-drug conjugates are considered promising cancer therapeutics.

[0004] ADCs have a more complex heterostructure than antibodies, posing an even greater challenge to their formulation for therapeutic use. Summary of the Invention

[0005] The present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate and a buffering agent, wherein the antibody-drug conjugate has the structure shown in Formula I: [ka] Among them, Ab is Pertuzumab n is 1 to 10, The buffer is selected from a citrate buffer, a histidine buffer, and a succinate buffer.

[0006] In some embodiments, the buffering agent is selected from histidine-histidine hydrochloride, citric acid-sodium citrate, and succinic acid-sodium succinate.

[0007] In some embodiments, the range of drug loading (n) may be the average number of cytotoxic drug conjugates per Pertuzumab antibody. Non-limiting examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and any range between these values. For example, the average number of cytotoxic drug conjugates per antibody may be 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 4, 3 to 5, 3.5 to 4.7, 5 to 6, 5 to 7, 5 to 8, and 6 to 8. Illustratively, drug loading (n) may be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. n is a decimal or an integer. In some embodiments, n is 1 to 8 or 3 to 5.

[0008] In some embodiments, the pharmaceutical composition has a pH of 3.5 to 5.5, including, but not limited to, 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 therebetween. In some embodiments, the pH is 3.5 to 5.0, or 3.7 to 4.7.

[0009] In some embodiments, the concentration of the buffering agent 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 values; in some embodiments, the concentration of the buffering agent is 10 mM to 50 mM; in some embodiments, the concentration of the buffering agent is 20 mM to 40 mM; and in some embodiments, the concentration of the buffering agent is 30 mM.

[0010] In some embodiments, the pharmaceutical composition further comprises a surfactant, such as polysorbate, poloxamer, Triton, sodium dodecylsulfonate, sodium laurylsulfonate, sodium octylglucoside, laurylsulfobetaine, myristylsulfobetaine, linolesulfobetaine, stearic-sulfobetaine, laurylsarcosine, myristylsarcosine, linolesarcosine, stearic-sarcosine, linolesbetaine, myristylbetaine, cetylbetaine, lauramidopropylbetaine, or cocamide. The surfactant may be selected from propyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol, etc. In some embodiments, the surfactant is a poloxamer or polysorbate, such as poloxamer 188, polysorbate 20, or polysorbate 80.

[0011] In some embodiments, 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, and 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 point values.

[0012] In some embodiments, the pharmaceutical composition further comprises a sugar. "Sugar" in the present disclosure refers to sugars of the general composition (CHO). nand derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The sugar may be selected from glucose, sucrose, trehalose, α,α-trehalose dihydrate, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc. In some embodiments, the sugar is sucrose.

[0013] In some embodiments, the concentration of sugar in the pharmaceutical composition is 25 mg / mL to 80 mg / mL, preferably 30 mg / mL to 50 mg / mL, including, but not limited to, 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 values. In some embodiments, the concentration is 40 mg / mL.

[0014] In some embodiments, the pharmaceutical composition further comprises an amino acid, such as glycine.

[0015] In some embodiments, the concentration of the amino acid in the 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, including, but not limited to, 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 values. In some embodiments, the concentration is 9 mg / mL.

[0016] In some embodiments, the concentration of the antibody-drug conjugate in the pharmaceutical composition is 1 mg / mL to 100 mg / mL in terms of 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, and 24 mg / mL. mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, and any range between these point values; in some embodiments, the concentration of the antibody-drug conjugate is 10 mg / mL to 30 mg / mL, for example 20 mg / mL, in terms of protein concentration. Specific, 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, 20.91 mg / mL, 20.92 mg / mL, 20.93 mg / mL, 20.94 mg / mL, 20.95 mg / mL, 20.96 mg / mL, 20.97 mg / mL, 20.98 mg / mL, 20.99 mg / mL, 21 mg / mL, and any range between these point values. The above "at protein concentration" means that the concentration is measured in terms of the antibody moiety in the antibody-drug conjugate.

[0017] In some embodiments, the pharmaceutical composition comprises: (a) the antibody-drug conjugate having a protein concentration of 1 mg / mL to 100 mg / mL; (b) 0.1 mg / mL to 0.8 mg / mL of poloxamer or polysorbate; (c) 30 mg / mL to 50 mg / mL sucrose; (d) 6 mg / mL to 15 mg / mL glycine, and (e) 10 mM to 50 mM succinic acid-sodium succinate buffer; The pH of the pharmaceutical composition is 3.5 to 5.5.

[0018] In some embodiments, the pharmaceutical composition comprises: (a) the antibody-drug conjugate having a protein concentration of 1 mg / mL to 50 mg / mL; (b) 0.2 mg / mL to 0.8 mg / mL of poloxamer or polysorbate; (c) 30 mg / mL to 50 mg / mL sucrose; (d) 6 mg / mL to 15 mg / mL glycine, and (e) 20 mM to 40 mM succinic acid-sodium succinate buffer; The pH of the pharmaceutical composition is 3.7 to 4.7.

[0019] In some embodiments, the pharmaceutical composition comprises: (a) the antibody-drug conjugate at a protein concentration of 20 mg / mL; (b) 0.6 mg / mL poloxamer 188 or 0.2 mg / mL polysorbate 80; (c) 40 mg / mL sucrose; (d) 9 mg / mL glycine, and (e) 30 mM succinic acid-sodium succinate buffer; The pH of the pharmaceutical composition is 4.2.

[0020] In some embodiments, any one of the pharmaceutical compositions is a liquid formulation. The liquid or reconstituted formulations of the present disclosure have relatively good stability. Furthermore, stable liquid formulations include liquid formulations that exhibit desirable properties after storage at temperatures including 40° C. for one month.

[0021] The present disclosure further provides a lyophilized formulation comprising an antibody-drug conjugate, the lyophilized formulation being capable of forming the pharmaceutical composition described above after reconstitution of the formulation.

[0022] The present disclosure further provides a method for preparing a lyophilized formulation comprising an antibody-drug conjugate, the method comprising the step of lyophilizing the pharmaceutical composition described above.

[0023] In some embodiments, 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.

[0024] The present disclosure further provides a lyophilized formulation comprising an antibody-drug conjugate, the lyophilized formulation being obtainable by lyophilizing the above-described pharmaceutical composition of the antibody-drug conjugate.

[0025] The present disclosure further provides a reconstitution solution comprising an antibody-drug conjugate, the reconstitution solution being prepared by reconstituting the above-described lyophilized formulation.

[0026] The present disclosure further provides a method for preparing the reconstitution solution, the method comprising the step of reconstituting the lyophilized formulation, wherein the solution used for the reconstitution is selected from, but not limited to, water for injection, saline, or glucose solution.

[0027] The present disclosure further provides an article of manufacture comprising a container containing the pharmaceutical composition, lyophilized formulation, or reconstitution solution. In some embodiments, the container is an injection bottle made of neutral borosilicate glass tubing.

[0028] The present disclosure further provides the use of the above pharmaceutical composition or lyophilized formulation or reconstituted solution or product in the preparation of a medicament for treating or preventing tumors.

[0029] The present disclosure further provides a method of treating a disease, comprising providing to a patient in need thereof the pharmaceutical composition or lyophilized formulation or reconstituted solution or product described above.

[0030] The present disclosure further provides the above pharmaceutical composition, or lyophilized formulation, or reconstituted solution, or product as a medicament for use in treating a disease.

[0031] The present disclosure further provides the above pharmaceutical composition, or lyophilized formulation, or reconstituted solution, or product as a medicament for use in treating tumors.

[0032] The present disclosure further provides the use of the above pharmaceutical composition, or lyophilized formulation, or reconstituted solution, or product as a medicament in the preparation of a medicament for treating tumors.

[0033] In some embodiments, the tumor is a cancer associated with expression of domain II of HER2.

[0034] In some embodiments, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric 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.

[0035] The heavy and light chain variable region sequences of Pertuzumab are as follows: Light chain variable region DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK SEQ ID NO: 1 Heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS SEQ ID NO: 2 Below is the sequence of Pertuzumab: Light chain DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 3 heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:4 As will be appreciated by those skilled in the art, one, some, or all of the features of each embodiment described in the present disclosure can be further combined to form other embodiments of the present disclosure. These and other combined embodiments of the present disclosure are further illustrated in the detailed description below.

[0036] The present disclosure provides pharmaceutical compositions that are more convenient to manufacture and administer and have stable performance. Among these, undesirable instability may include any one or more of 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, toxin release, etc. Specifically, the pharmaceutical compositions described in the present disclosure comprise an antibody-drug conjugate and a buffer.

[0037] term In order that this disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0038] An "antibody drug conjugate (ADC)" is an antibody linked via a linker unit to a biologically active cytotoxin or cell-killing small molecule drug.

[0039] The "drug loading" or "drug load," also known as the drug-to-antibody ratio (DAR), refers to the average number of drugs coupled to each antibody in an ADC. For example, it may be within the range of 1 to 10 drugs conjugated to each antibody. In some embodiments, it may be within the range of 1 to 8 drugs conjugated to each antibody, preferably 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 4, 3 to 5, 5 to 6, 5 to 7, 5 to 8, and 6 to 8. For example, the drug loading may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The general formula of an ADC according to the present disclosure includes a set of antibodies conjugated to drugs within the above range. In embodiments of the present disclosure, the drug loading may be denoted as n. The drug loading can be measured by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC.

[0040] The term "linker unit" or "linking fragment" or "linking unit" refers to a chemical structural fragment or bond that is linked at one end to an antibody or antigen-binding fragment thereof and at the other end to a drug, and may be linked to another linker before being linked to a drug.

[0041] The linker may comprise an extender, a spacer, and an amino acid unit and may be synthesized by methods known in the art, such as those described in US20050238649A1. The linker may be a "cleavable linker" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0042] The term "drug-linked arm fragment" or "drug-linker fragment" refers to a fragment in which a drug is linked to a linker unit, and can be linked to an antibody via the other end of the linker unit.

[0043] The loading of the cytotoxic drug is (1) controlling the molar ratio of the drug-linking arm fragment to the monoclonal antibody; (2) controlling the reaction time and temperature; (3) selecting different reaction reagents; The amount of oxygen can be controlled by a number of methods, including but not limited to:

[0044] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0045] The term "antibody" as used in this disclosure is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, full-length antibodies and antibody fragments (or antigen-binding fragments or antigen-binding portions), provided they exhibit the desired antigen-binding activity. Typically, naturally occurring intact antibodies are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0046] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. In one preferred conventional technique, a mammalian expression system induces antibody glycosylation, particularly at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium from which the antibodies are secreted can be purified by conventional techniques, for example, on an A or G Sepharose FF column containing a conditioned buffer. Nonspecifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and the antibody fragments are detected and collected by SDS-PAGE. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and multimers can be removed by conventional methods, such as molecular sieving or ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0047] "Buffer" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0048] A "histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, and histidine-histidine sulfate, with histidine-histidine hydrochloride being preferred. Histidine-histidine hydrochloride buffers may be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0049] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. A preferred citrate buffer is citric acid-sodium citrate.

[0050] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium salt, succinic acid-potassium succinate, succinic acid-calcium salt, etc. A preferred succinate buffer is succinic acid-sodium salt. Illustratively, the succinic acid-sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate salt.

[0051] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0052] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate.

[0053] A "pharmaceutical composition" refers to a mixture containing one or more antibody-drug conjugates described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof, together with other chemical components such as physiologically / pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to maintain the stability of the antibody active ingredient, facilitate administration to the body, and contribute to the absorption of the active ingredient to further exert its biological activity.

[0054] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0055] Unless otherwise specified, the solvent in any solution form of the pharmaceutical composition described in the present disclosure is water.

[0056] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a freeze-drying step is performed on a liquid or solution formulation.

[0057] As used herein, the terms "about" and "approximately" mean that a numerical value is within an acceptable error range of a specific value as determined by one of ordinary skill in the art, depending on how the numerical value is measured or determined (i.e., the limitations of the measurement system). For example, in the practice of the art, "about" may mean within or more than one standard deviation. Alternatively, "about" or "essentially comprising" may mean a range of at most 20%. Moreover, particularly for biological systems or processes, the terms may mean at most one order of magnitude or at most 5 times the numerical value. Unless otherwise indicated, when specific values ​​appear in this application and claims, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of the specific value.

[0058] The values ​​described in this disclosure are measured by an instrument or calculated after measuring by an instrument, and have a certain degree of error, generally within a reasonable error range of ±10%. Of course, the context in which the value is used should be taken into consideration. For example, in the case of the content of total impurities, the error of the value after measurement should be ±10% or less, and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, and preferably ±5%.

[0059] The pharmaceutical compositions described herein can achieve stable efficacy, i.e., the antibody-drug conjugate therein essentially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition essentially retains its physical and chemical stability and biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are several analytical techniques for measuring the stability of proteins or antibody-drug conjugates, which can be used to measure stability after storage at a predetermined temperature for a predetermined period of time.

[0060] A stable formulation is one that shows no significant change when stored at refrigerated temperatures (2°C to 8°C) for at least 3 months, preferably 6 months, and more preferably 1 year. Stable liquid formulations also include liquid formulations that exhibit desired characteristics after storage at temperatures including 25°C for periods including 1 month, 2 months, and 3 months. Stable liquid formulations also include liquid formulations that exhibit desired characteristics after storage at temperatures including 40°C for periods including 10 days, 20 days, and 1 month. Typical examples of stability include those measured by SEC-HPLC in which aggregation or degradation of antibody monomers typically occurs at no more than about 10%, preferably no more than about 5%. Visual analysis reveals that the formulation is a pale yellow, almost colorless, transparent liquid, or colorless, or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%. A decrease of no more than about 10%, preferably no more than about 5%, is typically observed. No more than about 10%, preferably no more than about 5%, of the antibody monomers typically forms aggregates.

[0061] An antibody-drug conjugate "retains its physical stability" in a drug formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation, as determined by visual 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 the tertiary structure of proteins) and FTIR spectroscopy (which determines the secondary structure of proteins).

[0062] An antibody-drug conjugate "retains its chemical stability" in a drug formulation if it does not undergo significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or cleavage (e.g., assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (e.g., assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (e.g., assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, or isoaspartic acid content measurement), and isomerization (e.g., assessed by isoaspartic acid content measurement, peptide mapping, etc.).

[0063] An antibody-drug conjugate "retains its biological activity" in a drug formulation if the biological activity at a given time is within a predetermined range of the biological activity exhibited when the drug formulation is prepared.

[0064] "Optional" or "optionally" means that the subsequently described event or circumstance may occur, but does not necessarily occur, and the description includes both cases where the event or circumstance occurs and cases where it does not. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but are not necessarily present.

[0065] The term "substituted" refers to one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, being independently replaced with a corresponding number of substituents. Of course, substituents are only located at their chemically feasible positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino group or a hydroxy group having free hydrogen may be unstable if it is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0066] The preparation of conventional pharmaceutical compositions is set out in the Chinese Pharmacopoeia.

[0067] The term "carrier," as used in the present disclosure, refers to a system that can change the drug's entry into and distribution within the human body, control the drug's release rate, and transport the drug to target organs. The release and targeting system of the drug carrier can reduce drug degradation and loss, lower side effects, and improve bioavailability. For example, polymer surfactants, which are used as carriers, can self-assemble to form various types of aggregates due to their unique amphiphilic structure, and preferred examples include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and good membrane permeability, making them good drug carriers.

[0068] "Administration" and "treatment," when applied to an animal, human, or experimental subject, cell, tissue, organ, or biological fluid, refer to contact of an exogenous agent, therapeutic agent, or diagnostic agent or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with the cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Administration" and "treatment" also refer to ex vivo and in vitro treatment, e.g., of cells, with a reagent, diagnostic, binding composition, or via another cell. "Treatment," when applied to a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, and research and diagnostic uses.

[0069] "Treatment" refers to administering to a patient, for example, an oral or topical therapeutic agent comprising a composition of any one of the binding compounds disclosed herein, wherein the patient has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is administered an amount of therapeutic agent that effectively alleviates one or more disease symptoms, thereby inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on various factors, including the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. Although embodiments of the present disclosure (e.g., methods of treatment or products) may be ineffective in alleviating their respective target disease symptoms, any statistical testing method known in the art, such as Student's t-test, chi-squared test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, will confirm that they will reduce the target disease symptoms in a statistically significant number of patients.

[0070] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. Effective amount further refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as the condition being treated, the patient's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0071] "Replacement" refers to the replacement of a solvent system that dissolves the antibody protein or antibody-drug conjugate, for example, replacing a high-salt or high-osmolarity solvent system containing the antibody protein or antibody-drug conjugate with a buffer system of a stable formulation by a physical manipulation method such that the antibody protein or antibody-drug conjugate is present in a stable formulation. Such physical manipulation methods include, but are not limited to, ultrafiltration, dialysis, or redissolution after centrifugation.

[0072] As used herein, "Pertuzumab" refers to an antibody comprising the light chain and heavy chain variable region amino acid sequences in SEQ ID NOs: 1 and 2, respectively. When Pertuzumab is a complete antibody, it preferably comprises the light chain and heavy chain amino acid sequences in SEQ ID NOs: 3 and 4, respectively. [Brief explanation of the drawings]

[0073] [Figure 1] FIG. 1 shows a graph of changes in tumor volume in N87 / 16-8 subcutaneously transplanted tumor model mice. [Figure 2] FIG. 1 shows a graph of body weight changes in N87 / 16-8 subcutaneously transplanted tumor model mice. [Figure 3] FIG. 1 shows a graph of changes in tumor volume in a JIMIT-1 subcutaneously transplanted tumor model mouse. [Figure 4] FIG. 10 is a graph showing changes in body weight of JIMIT-1 subcutaneously transplanted tumor model mice. DETAILED DESCRIPTION OF THE INVENTION

[0074] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. In the examples of the present disclosure, experimental methods for which specific conditions are not specified generally follow standard conditions, such as those in the "Antibody Technology Laboratory Manual" and "Molecular Cloning Manual" published by Cold Spring Harbor Laboratory, or the conditions recommended by raw material or product manufacturers. Reagents for which specific sources are not specified are standard commercially available reagents.

[0075] The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts (δ) are 10 -6 The values ​​are shown in units of ppm. NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD) as the measurement solvents, and tetramethylsilane (TMS) as the internal standard.

[0076] For MS measurements, a liquid chromatograph mass spectrometer Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS (manufacturer: Agilent, MS model number: 6110 / 6120 Quadrupole MS) was used.

[0077] Waters ACQuity UPLC-QD / SQD (manufacturer: waters, MS model number: waters ACQuity Qda Detector / waters SQ Detector) and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model number: THERMO Q Exactive) were used.

[0078] High-performance liquid chromatography (HPLC) analysis was performed using high-pressure liquid chromatographs Agilent HPLC 1200DAD, Agilent HPLC 1200VWD, and Waters HPLC e2695-2489.

[0079] For chiral HPLC analysis, a high performance liquid chromatograph Agilent 1260 DAD was used.

[0080] For high-performance liquid preparative chromatography, preparative chromatographs Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 were used.

[0081] For chiral separation, a preparative chromatograph Shimadzu LC-20AP was used.

[0082] Combiflash Rf200 (TELEDYNE ISCO) was used as the CombiFlash high-speed preparative chromatograph.

[0083] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used as silica gel plates for thin layer chromatography. The specifications of the silica gel plates used for thin layer chromatography (TLC) are 0.15-0.2 mm, and the specifications for separating and purifying products by thin layer chromatography are 0.4-0.5 mm.

[0084] For silica gel column chromatography, 200-300 mesh silica gel manufactured by Yantai Huanghai Silica Gel was generally used as the carrier.

[0085] Known starting materials according to the present disclosure may be synthesized by or according to methods known in the art, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0086] In the examples, unless otherwise specified, all reactions can be carried out under an argon or nitrogen gas atmosphere.

[0087] The argon or nitrogen gas atmosphere refers to an argon or nitrogen gas balloon with a volume of about 1 L connected to the reaction flask.

[0088] The hydrogen gas atmosphere refers to a hydrogen gas balloon with a volume of approximately 1 L connected to the reaction flask.

[0089] For the pressurized hydrogenation reaction, a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen gas generator or an HC2-SS hydrogenation apparatus were used.

[0090] The hydrogenation reaction was usually carried out by repeating the procedure of evacuating and filling with hydrogen gas three times.

[0091] A CEM Discover-S 908860 microwave reactor was used for the microwave reactions.

[0092] In the examples, unless otherwise specified, the solution refers to an aqueous solution.

[0093] In the examples, unless otherwise specified, the reaction temperature is room temperature, 20 to 30°C.

[0094] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used to purify the compound include A: dichloromethane and isopropyl alcohol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system, and the volume ratio of the solvents may be adjusted depending on the polarity of the compound, or by adding a small amount of triethylamine and an acidic or basic reagent, etc.

[0095] The antibody drug conjugates of Formula I described herein can be prepared according to the methods of PCT / CN2022 / 107479.

[0096] 1. Preparation of antibody conjugates Example 1 [ka] Step 1: Preparation of Compound 1b Compound 1a (eribulin, prepared according to the method of ZL201010236637.2) (72.91 mg, 0.1 mmol) was dissolved in 10 mL of tetrahydrofuran in an ice-water bath, and Fmoc-OSu (fluorenylmethyl succinimidyl carbonate, 41 mg, 0.12 mmol) was added. The mixture was stirred at room temperature until the reaction was complete. The crude product was obtained by concentrating under reduced pressure and used directly in the next reaction.

[0097] Step 2: Preparation of compound 1c The crude product of 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. After filtration, the mixture was concentrated under reduced pressure to obtain the crude product, which was then used directly for the next reaction.

[0098] Step 3: Preparation of Compound 1 The crude product of 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 then concentrated under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / ethyl acetate / petroleum ether) to obtain 3 mg of the target product, compound 1.

[0099] LC / MS (ESI): m / z 744.2 [M+H] + .

[0100] [ka] In an ice-water bath, 4a (50 mg, 0.08 mmol, prepared according to the method in WO2017151979) was dissolved in 1.5 mL of N,N-dimethylformamide, and then DIPEA (N,N-diisopropylethylamine, 18 mg, 0.14 mmol) was added, followed by the addition of bis(p-nitrophenyl)carbonate (49 mg, 0.16 mmol). The mixture was stirred at room temperature, and 20 mL of methyl tert-butyl ether was added. The mixture was stirred for 20 minutes, filtered, and dried to obtain 36 mg of a solid. LC / MS (ESI): m / z 784.1 [M+H] + It was.

[0101] [ka] Compound 1 (13.5 mg, 0.018 mmol) was dissolved in 1.5 mL of DMF, DIPEA (7 mg, 0.054 mmol) was added, and then compound 4b (18 mg, 1.3 mmol) was added in several portions. The mixture was stirred until the reaction was almost complete, and concentrated to obtain the crude product. This was separated by HPLC preparative separation to obtain 6.5 mg of compound L-1, with a purity of 96.95%. LC / MS (ESI): m / z 1388.3 [M+H] + It was.

[0102] Example 2 ADC-001 [ka] At 37°C, the prepared tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) aqueous solution (10 mM, 24.2 μL, 242 nmol) was added to a PBS buffer solution of the antibody Pertuzumab (0.05 M PBS buffer solution with pH = 6.5, 10.0 mg / mL, 1.43 mL, 97 nmol), placed in a water bath shaker, and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0103] Compound L-1 (1.34 mg, 965 nmol) was dissolved in 60 μL of dimethyl sulfoxide and added to the reaction mixture. The mixture was placed in a water bath shaker and reacted at 25 °C for 3 hours with shaking, after which the reaction was stopped. The reaction mixture was desalted and purified on a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain the title product in PBS buffer (1.24 mg / mL, 9.8 mL). This product was then stored refrigerated at 4 °C.

[0104] The mean value was calculated using CE-SDS: n = 3.17.

[0105] Example 3 ADC-001 At 12°C, 135.32 g of Pertuzumab antibody (0.93 mmol) was reacted with 0.5880 g of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 2.05 mmol) in 20 mM histidine-HCl-Tris buffer (pH 7.2, 7.73 L) containing 2.5 mM EDTA in a constant temperature water bath with stirring for 120 minutes to obtain a solution of intermediate I. The pH of the reaction solution was adjusted to 5.2 by dropwise addition of 2 M acetic acid.

[0106] Compound L-1 (6.865 g, 4.94 mmol) was dissolved in 0.425 L of DMSO to obtain a DMSO solution of compound L-1. 0.348 L of DMSO was added to the intermediate I solution, and the DMSO solution of compound L-1 was added to the intermediate I solution to which DMSO had been added, followed by stirring in a water bath at 12°C. 2 M acetic acid was added dropwise to adjust the pH of the reaction solution to 4.2.

[0107] The reaction solution was filtered through a 0.22 μm filter and then ultrafiltered using succinate buffer (pH = 4.2) to obtain the product ADC-001 (20 g / L, mean value calculated by reverse phase chromatography: n = 4.1).

[0108] 2. Biological testing Test Example 1: Inhibitory effect on the proliferation of in vitro cultured human breast cancer BT-474 and human gastric cancer NCI-N87, NCI-N87 / 16-8, and NCI-N87 / 8-2 cells 1. Drug Information [Table 1] Note: The above samples were provided by Shanghai Hengrui Co., Ltd. Here, pertuzumab was prepared according to a similar method in WO2006033700 and is a kadcyla:trastuzumab-emtansine conjugate.

[0109] 1.2 Test cells NCI-N87 and BT-474 cells were purchased from the American Type Culture Collection (ATCC). T-DM1-resistant NCI-N87 / 16-8 and NCI-N87 / 8-2 cells were constructed in our laboratory by long-term treatment with T-DM1 on NCI-N87. Cells were cultured in RPMI 1640 / DMEM (1:1) medium containing 10% fetal bovine serum (FBS).

[0110] 1.3 Equipment and Reagents RPMI 1640 and DMEM were purchased from Gibco BRL, FBS from Gibco, and sulforhodamine B (SRB) from Sigma.

[0111] The microplate reader Synergy H4 was purchased from BioTek.

[0112] 1.4 Experimental Procedure A fixed number of cells in the logarithmic growth phase were seeded onto a 96-well culture plate. After 24 hours of adherent growth, various concentrations of drugs (10,000, 3,000, 1,000, 300, 100, 30, 10, 3, and 1 ng / mL) were added. After 120 hours of drug treatment, the cells were fixed with trichloroacetic acid, stained with SRB solution, and finally, Tris solution was added to dissolve the SRB. The OD value was measured at a wavelength of 510 nm using a microplate reader, and the cell growth inhibition rate was calculated using the following formula: Inhibition rate = (OD value control well - OD value treatment well) / OD value control well x 100% The median inhibitory concentration (IC) was calculated using the software GraphPad Prism 7 at each inhibitor concentration. 50 was calculated.

[0113] [Table 2]

[0114] Test Example 2: In vitro cytotoxic activity screening of Compound 1 1.1. Experimental principles and methods In this experiment, CTG was used to detect ATP content, which reflected the survival status of tumor cells. First, cells were seeded at different densities and cultured for 3 and 5 days, and then IC 50 The final culture conditions were determined based on the maximum inhibition rate and the maximum inhibition rate, and the killing effect of the toxin molecule was detected under these conditions.

[0115] 1.2. Cell line selection Two disease models, breast cancer and NSCLC, were selected based on the experimental objectives, and three cell lines, SKBR3 tumor cells (HER2+, ATCC, product number HTB-30), MDA-MB-468 (HER2-, ATCC, product number HTB-132), and A549 (human non-small cell lung cancer cells, ATCC, product number CCL-185), were selected for screening experiments.

[0116] 1.3. Determining cell culture conditions 1) Cell culture: A549, SK-BR-3, and MDA-MB-468 cells were cultured in Ham's F-12K (Kaighn's) medium (Gibco, 21127030) containing 10% FBS (Gibco, 10099-141), McCoy's 5A medium (ThermoFisher, product number 16600108), and Leibovitz's L-15 medium (ThermoFisher, product number 11415-114), respectively.

[0117] 2) Cell seeding: A549 cells were digested with trypsin, then suspended in the above medium, counted, and seeded with 4.3 × 10 5, 7.2 × 10 5 , 11.5 x 10 5 Cells were taken from each well and medium was added to bring the final volume to 26 mL. 180 μL of cell suspension was added to each well in rows 2 to 11 of a 96-well plate (Corning, Product No. 3903) to achieve cell densities of 3K, 5K, and 8K, respectively. 200 μL of medium was added to row 12, and the remaining wells were filled with PBS. The above procedure was repeated for SKBR3 and MDA-MB-468 cells. Two parallel samples were prepared.

[0118] 3) Drug Preparation: Stock solutions of the positive control eribulin and the compounds of the present disclosure were prepared in DMSO in a round-bottom 96-well plate (Corning, Product No. 3788). A 2 mM stock solution (10-fold dilution of the stock solution with DMSO) was prepared in column 1 of Dispensing Plate 1, and then diluted 10-fold with DMSO up to column 10, with column 11 being DMSO. 95 μL of culture medium was added to each well in columns 2 to 11 of Dispensing Plate 2, and 5 μL of the solution was aspirated from columns 2 to 11 of Dispensing Plate 1 and added to Dispensing Plate 2. After uniform mixing, 20 μL was aspirated and added to the seeded cells. The cells were cultured for 3 and 5 days.

[0119] 4) CTG detection (Cell Titer-Glo™, luminescent cell viability assay, Promega): On the third and fifth days, the cell plates were removed and equilibrated to room temperature. 90 μL of CTG was added to each well, and the reaction was allowed to proceed in the dark at room temperature for 10 minutes. The luminescence was then read using a microplate reader to determine the IC. 50 was calculated.

[0120] 1.4. Data Results [Table 3]

[0121] Test Example 3: Pharmacokinetics test of Compound 1 1. Overview Non-naive beagle dogs were used as test animals, and the plasma drug concentrations were measured at various time points after intravenous injection of Compound 1 and eribulin using LC / MS / MS. The pharmacokinetic behavior of the compound according to the present disclosure in dogs was studied, and its pharmacokinetic characteristics were evaluated.

[0122] 2. Test plan 2.1 Study Drug Compound 1 and eribulin 2.2 Test animals Six male beagle dogs purchased from Medicilon Preclinical Research (Shanghai) LLC were divided into two groups on average and subjected to animal administration experiments.

[0123] 2.3 Drug Preparation Compound 1 was weighed and dissolved in 5% by volume of DMSO, 20% by volume of PG and 20% by volume of PEG400, and 55% by volume of saline was added to prepare a 0.25 mg / mL colorless and transparent solution.

[0124] Eribulin was weighed and dissolved in 5% by volume of DMSO, 20% PG, and 20% PEG400, and 55% saline was added to prepare a 0.25 mg / mL clear and colorless solution.

[0125] 2.4 Administration Dogs in one group were administered Compound 1 by intravenous injection, with the dose being 0.5 mg / kg in all cases and the administration volume being 2 mL / kg in all cases.

[0126] Dogs in the other group received eribulin intravenously, with all doses at 0.5 mg / kg and all dose volumes at 2 mL / kg.

[0127] 3, operation Dogs were injected with Compound 1, and 1 mL of blood was collected before administration, 5 minutes after administration, and 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours later. The collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice. Plasma was centrifuged within 1 hour (centrifugation at 2200 g for 10 minutes at 2-8°C). Plasma samples were stored in a -80°C freezer until testing.

[0128] Dogs were administered an eribulin compound by injection, and 1 mL of blood was collected before administration, 5 minutes after administration, and 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0 hours later. The collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, and the collected whole blood was placed on ice. Plasma was centrifuged within 1 hour (centrifugation at 2200 g for 10 minutes at 2-8°C). Plasma samples were stored in a -80°C freezer until testing.

[0129] After drug administration, the content of compounds to be measured in dog plasma was measured as follows: 25 μL of dog plasma was collected at each post-administration time point, and 50 μL (100 ng / mL) of camptothecin (China Institute for Biological Products Testing) internal standard solution and 200 μL of acetonitrile were added. The plasma samples were vortex-mixed for 5 minutes and centrifuged for 10 minutes (3700 rpm). 3-4 μL of the supernatant was then analyzed by LC / MS / MS (API4000 triple quadrupole tandem mass spectrometer (No. 2), Applied Biosystems, USA; Shimadzu LC-30AD ultra-high performance liquid chromatography system, Shimadzu, Japan).

[0130] 4. Pharmacokinetic parameter results The pharmacokinetic parameters of the compounds of the present disclosure are shown in Table 3 below.

[0131] [Table 4]

[0132] Test Example 4: Therapeutic effect of ADC-001 on human gastric cancer NCI-N87 / 16-8 tumors subcutaneously transplanted into nude mice 1. Purpose of the experiment To evaluate the therapeutic effect of ADC-001 on human gastric cancer NCI-N87 / 16-8 tumors subcutaneously transplanted into nude mice.

[0133] 2. Test drug ADC-001 diluted to the desired concentration in saline.

[0134] 3, cells Human gastric cancer NCI-N87 cells were purchased from the American Type Culture Collection. NCI-N87 cells exhibited resistance to T-DM1 during long-term induction culture with T-DM1 and were designated NCI-N87 / 16-8. Cells were cultured in 10-cm culture dishes in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged two to three times weekly. When the cells reached exponential growth phase, they were digested with trypsin, harvested, counted, and inoculated.

[0135] 4. Test animals Four-week-old female BALB / cJGpt-Foxn1 mice were purchased from Jiangsu Jisu Yaokang Biotechnology Co., Ltd. nu / Gpt mice. Production permit number: SCXK (Su) 2019-0009, Animal Certification Number: 202004958. Rearing environment: SPF grade.

[0136] 5. Test procedure 1 × 10 for each nude mouse 7 NCI-N87 / 16-8 cells were subcutaneously inoculated, and tumors were 100-150 mm 3 Once the tumors reached the tumor size, the animals were divided into groups based on tumor volume and body weight (D0). Mice were intravenously administered 0.3 and 0.6 mg / mL of ADC-001 at a dose volume of 10 mL / kg. Tumor volume was measured twice a week, and the mice were weighed and the data were recorded.

[0137] 6. Test indicators The experimental indicators are for examining the effect of the drug on tumor growth, and the specific indicators are T / C% or tumor growth inhibition rate (TGI%).

[0138] The diameter of the tumor was measured twice a week with a caliper, and the calculation formula for the tumor volume (V) is as follows: V = 1 / 2 × a × b 2 Among them, a and b represent the length and width respectively.

[0139] T / C(%) = (T - T0) / (C - C0) × 100. Among them, T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0140] Growth inhibition rate TGI(%) = 100 - T / C(%).

[0141] When tumor regression appears, growth inhibition rate %(TGI%) = 100 - (T - T0) / T0 × 100 When the tumor becomes smaller than the initial volume, that is, when T < T0 or C < C0, it is defined as partial regression (PR) of the tumor, and when the tumor completely disappears, it is defined as complete regression (CR) of the tumor.

[0142] When the experiment ended, or the end point of the experiment was reached, or the average tumor volume of the solvent group reached 1500 mm3, the animals were killed by CO2 anesthesia, then dissected, and the tumors were removed and photographed.

[0143] 7. Statistical analysis Unless otherwise specified, for the comparison of tumor volumes between the two groups, two-sided Student's t-test is adopted, and when P < 0.05, it is defined that there is a statistically significant difference.

[0144] 8. Results On day 22 after a single dose of 3 mg / kg and 6 mg / kg, ADC-001 inhibited the growth of NCI-N87 / 16-8 cells in a nude mouse subcutaneous tumor model, with tumor inhibition rates of 46.77% and 90.18%, respectively (P<0.01 vs. PBS control). Tumor-bearing mice tolerated ADC-001 well and did not experience any symptoms such as weight loss.

[0145] 9, Conclusion ADC-001 can effectively inhibit the growth of human gastric cancer NCI-N87 / 16-8 cells in a nude mouse subcutaneous tumor model, and ADC-001 is well tolerated by tumor-bearing mice.

[0146] Test Example 5: Therapeutic effect of ADC-001 on JIMIT-1 cells in subcutaneously transplanted tumors in nude mice 1. Test drug ADC-001 diluted to the desired concentration in PBS.

[0147] 2, cells Human breast cancer JIMIT-1 cells.

[0148] 3. Test animals Female Balb / c nude mice 4. Test procedure BALB / c nude mice were inoculated with 5 × 10 JIMT1 cells into the right dorsal region. 5 JIMT1 cells were subcutaneously inoculated, and tumor volumes were 80–100 mm 3 The mice were randomly divided into groups of six mice each based on tumor volume and body weight, and treatment began on the day of grouping. HRA00092-C063-004 was diluted to 0.6 mg / mL in PBS and administered intravenously at a dose of 10 mL / kg. The control group received the same volume of PBS intravenously.

[0149] The major and minor diameters of the tumors were measured twice a week with a vernier caliper, and the mice were weighed.

[0150] The formula for calculating tumor volume (V) is as follows: V=1 / 2×a×b 2 In this case, a and b represent the length and width, respectively.

[0151] T / C (%) = (T - T0 / (C - C0) × 100, where T and C are the tumor volumes at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0152] Growth inhibition rate TGI(%)=100-T / C(%).

[0153] Unless otherwise stated, two-tailed Student's T-test was used to compare tumor volumes between two groups, and a P value of <0.05 was defined as a statistically significant difference.

[0154] 5, results On the 24th day after a single administration, ADC-001 significantly inhibited the proliferation of JIMIT-1 cells in a nude mouse subcutaneous tumor model, with a tumor inhibition rate of 121.3%, of which 5 mice showed tumor regression (<50 mm). 3 Tumor-bearing mice tolerated ADC-001 well and did not experience symptoms such as weight loss.

[0155] 3. Preparations The equipment used during the preparation and detection of the formulation and the calculation method of the results are as follows:

[0156] SEC molecular exclusion chromatography: This is an analytical method that separates solutes based on the correlation between the pore size of gel pores and the coil size of polymer sample molecules.

[0157] SEC% (percentage of SEC monomer content) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas). ΔSEC% = SEC% of the formulation before stabilization - SEC% of the formulation after stabilization.

[0158] SEC measurement equipment: Agilent 1260, column: water, XBrige BEH200Å SEC (300 × 7.8 mm 3.5 μm) R-CE capillary gel electrophoresis: This is electrophoresis, which is carried out by moving a gel as a support medium in a capillary, and is a method of separating samples according to their molecular weight at a constant voltage.

[0159] R-CE% = A main peak / A total × 100% (A main peak is the peak area of ​​the light chain main peak + heavy chain main peak in the sample, and A total is the sum of all peak areas. ΔR-CE% = R-CE% of the formulation before stable storage - R-CE% of the formulation after stable storage. CE measuring equipment: Beckman model number plus800

[0160] Osmolality measurement: Osmotic pressure was measured using the freezing point method, and based on the fact that the freezing point depression value is directly proportional to the molar concentration of the solution, a highly sensitive temperature measuring element was used to measure the freezing point of the solution and convert it into osmotic pressure using an electrical quantity. The instrument manufacturer is Loser, and the model number is OM815.

[0161] Protein concentration: The protein used in the following examples is an anti-Her2-ADC.

[0162] Equipment for measuring protein concentration: UV-visible spectrophotometer, model number: Nano Drop 2000, light path 1 mm.

[0163] The concentration of the antibody drug conjugate in this disclosure is measured in terms of protein concentration, i.e., the concentration of the antibody moiety in the antibody drug conjugate.

[0164] The toxin in the antibody-drug conjugate is hardly absorbed at the protein characteristic absorption wavelength of 280 nm, and at the same time, the toxin is hardly absorbed at 370 nm either. Therefore, the concentration of the protein is calculated using the following formula:

number

[0165] When the test solution is diluted, the protein concentration

number

[0166] Measurement of free toxin (RP-UPLC reverse phase method): Proteins in the sample were removed by ACN precipitation, and the supernatant was removed, dried by nitrogen blowing, and redissolved. The toxins in the sample were separated based on their polarity, and the toxin content in the sample was calculated using a linear equation fitting the toxin standard concentration to the peak area.

[0167] Measuring equipment: Waters ACQuity H class, Column: Waters ACQUITY UPLC BEH Shield RP18, 1.7 μm, 2.1 x 150 mm

[0168] Measurement of free toxin (LC-MS method): This study quantitatively measured toxins and related impurities in ADC products using liquid chromatography-mass spectrometry. Samples were diluted and directly injected into a UPLC-MS for detection. Simultaneously, the free toxin content in the sample was measured by external standardization, comparing it with the chromatogram peak area of ​​the extracted ion flow of a toxin standard.

[0169] Equipment: Waters, Acquity UPLC_Rda (Bioaccord) Column: Waters, ACQUITY UPLC BEH C4, 1.7 μm, 2.1 x 50 mm

[0170] Formulation Example 1. pH and Buffer System Screening Formulations were prepared containing 20 mg / mL protein, 0.2 mg / mL polysorbate 80 (PS80), 40 mg / mL sucrose, and 9 mg / mL glycine in the buffer system shown in Table 4. Forced degradation studies were performed on the samples (40°C for 1 month) to investigate the effect of different buffer systems on protein stability using SEC as an evaluation index.

[0171] The results are shown in Table 4. The SEC data show that after 1 month at 40°C, formulations containing His-HCl or SA showed less loss of SEC monomer than the CA group. The His-HCl and SA systems are preferred.

[0172] [Table 5] NOTE: His-HCl represents histidine-histidine hydrochloride, CA represents citric acid-citric acid sodium salt, SA represents succinic acid-sodium succinate salt, 40 °C M1 represents leaving at 40 °C for 1 month, ΔSEC% represents the difference between D0 and 40 °C M1 SEC monomer, and so on hereafter.

[0173] Formulation Example 2. Effect of pH of the buffer system on free toxin A formulation containing 20 mg / mL of protein, 0.2 mg / mL of PS80, 40 mg / mL of sucrose, and 9 mg / mL of glycine was prepared using the buffer system shown in Table 5. The stability of the sample was examined at 2 to 8°C, and the effect of different pH values ​​on the free toxin was investigated.

[0174] The results are shown in Table 5. The free toxin data show that after 1 month at 2-8°C, the free toxin decreased with decreasing pH of the buffer system, with pH 5.5 being superior to pH 6.5.

[0175] [Table 6] Note: 2-8°C M1 indicates incubation at 2-8°C for one month.

[0176] Formulation Example 3. Effect of pH and buffer system on free toxin A formulation containing 20 mg / mL of protein, 0.2 mg / mL of polysorbate 80 (PS80), 40 mg / mL of sucrose, and 9 mg / mL of glycine was prepared using the buffer system shown in Table 6. The stability of the sample was examined (left at room temperature for one day, left at 2-8°C for one day, and then repeated two freeze-thaw cycles between -35°C and 2-8°C). The effect of differences in pH and buffer system on free toxin was investigated using free toxin as an evaluation index.

[0177] The results are shown in Table 6. The free toxin data indicate that the rate of increase of free toxin slows as the pH decreases, and that the buffer system SA is superior to His-AA. Therefore, the preferred buffer system is 30 mM SA pH 4.2.

[0178] [Table 7] Note: RT24H means leaving the sample at room temperature for one day, 2-8°C D1 means leaving the sample at 2-8°C for one day, FT2C means two cycles of freezing and thawing between -35°C and 2-8°C, and △free toxin means the increase in the amount of free toxin after leaving the sample compared to the value at time 0, and so on.

[0179] Formulation Example 4. Optimization of the pH of the buffer system A liquid formulation and a lyophilized powder formulation were prepared 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. The stability of the samples was examined (liquid samples were left at room temperature for 24 hours, at 2-8°C for 3 days, and subjected to five freeze-thaw cycles between -35°C and 2-8°C; lyophilized powder samples were left at 40°C for 4 weeks). The effects of different buffer pHs on protein stability and free toxins were investigated using appearance, SEC, R-CE, and free toxins as evaluation indices.

[0180] The results are shown in Tables 7 and 8. The results show that the solutions at pH 4.0-4.2 had relatively good SEC / CE / free toxin stability under each condition. Although the free toxin RT24h and 2-8°C D3 at pH 4.6 increased slightly, the free toxin remained low overall, indicating acceptable stability. The lyophilized formulations had good stability after being stored at pH 4.0-4.6 and 40°C for 4 weeks, with no difference between groups. Summarizing the solution and lyophilization results, the buffer system is preferably 30 mM SA pH 4.2.

[0181] [Table 8] Note: D0 represents 0 days, RT24H represents 24 hours at room temperature, D3 represents 3 days, and so on. FT1C represents one cycle of freeze-thawing between -35°C and 2-8°C, and so on.

[0182] [Table 9] Note: FR refers to reconstitution of lyophilized powder with water for injection, D0 refers to 0 days, and W4 refers to 4 weeks.

[0183] Formulation Example 5. Screening of excipients and osmolality adjusters Solutions containing the excipients and osmolality adjusters shown in Table 9, 30 mM SA pH 4.2, 20 mg / mL protein, and 0.2 mg / mL PS80 were prepared. Stability of the samples was evaluated to determine the effect of different concentrations of excipients and osmolality adjusters on protein stability using appearance, SEC, and free toxin as evaluation indices.

[0184] The results are shown in Table 10. RT24H / 2-8°C D1 / FT1C data showed no significant differences in appearance or SEC between groups. However, the 40 mg / mL sucrose + 9 mg / mL glycinate group had slightly better free toxins, and therefore, 40 mg / mL sucrose + 9 mg / mL glycine was preferred.

[0185] [Table 10]

[0186] [Table 11] NOTE: RT24H means leaving the sample at room temperature for 24 hours, 2-8°C D1 means leaving the sample at 2-8°C for one day, FT1C means repeating one freeze-thaw cycle between -35°C and 2-8°C, and N / A means not detected.

[0187] Formulation Example 6. Surfactant Screening A liquid formulation 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 left to stand (at room temperature for 7 days and at 4°C for 7 days), and the effect of different surfactants on protein stability was investigated using SEC and free toxin as evaluation indices.

[0188] The results are shown in Table 11. The data showed that there were no significant differences in free toxin or SEC purity between groups.

[0189] [Table 12]

[0190] Formulation Example 7. Surfactant concentration screening Liquid and lyophilized powder formulations containing F68 at different concentrations, 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 examined (solution samples were left at 2-8°C for 3 days, and freeze-thawed three times between -35°C and 2-8°C, and the freeze-dried products were left for 0 hours). The effect of different concentrations of F68 on protein stability was investigated using appearance and SEC as evaluation indices.

[0191] The results are shown in Table 12. The data showed that for liquid formulations, formulations containing 0.4 to 0.6 mg / mL of F68 had better sample appearance under different conditions, and there was no significant difference in SEC purity. There was no significant difference in appearance or SEC purity after lyophilization and reconstitution. Therefore, the surfactant concentration is preferably 0.4 to 0.6 mg / mL, and more preferably 0.6 mg / mL.

[0192] [Table 13]

Claims

1. 1. A pharmaceutical composition comprising an antibody-drug conjugate and a buffering agent, wherein the antibody-drug conjugate has the structure shown in Formula I: 【Chemistry 1】 Among them, Ab is Pertuzumab, n is 1 to 10, preferably 1 to 8, more preferably 3 to 5; The buffer is selected from citrate buffers, histidine buffers and succinate buffers, and is preferably histidine-histidine hydrochloride, citric acid-sodium citrate and succinic acid-sodium succinate. Pharmaceutical compositions.

2. The pharmaceutical composition according to claim 1, wherein the pH of the pharmaceutical composition is 3.5 to 5.5, preferably 3.5 to 5.0, more preferably 3.7 to 4.

7.

3. 2. The pharmaceutical composition according to claim 1, wherein the concentration of the buffering agent is 5 mM to 50 mM, preferably 20 mM to 40 mM, more preferably 30 mM.

4. 3. The pharmaceutical composition according to claim 1 or 2, further comprising a surfactant, said surfactant being preferably a polysorbate or a poloxamer.

5. 5. The pharmaceutical composition according to claim 4, wherein the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL, preferably 0.05 mg / mL to 1.0 mg / mL, more preferably 0.1 mg / mL to 1.0 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, further comprising a sugar, preferably sucrose.

7. 7. The pharmaceutical composition according to claim 6, wherein the concentration of the sugar is from 25 mg / mL to 80 mg / mL, preferably from 30 mg / mL to 50 mg / mL, more preferably 40 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, further comprising an amino acid, preferably glycine.

9. 9. The pharmaceutical composition according to claim 8, wherein the concentration of the amino acid is 6 mg / mL to 15 mg / mL, preferably 7 mg / mL to 11 mg / mL, more preferably 9 mg / mL.

10. the antibody-drug conjugate has a protein concentration of 1 mg / mL to 100 mg / mL; Preferably, the antibody-drug conjugate has a protein concentration of 10 mg / mL to 30 mg / mL; More preferably, the antibody-drug conjugate has a protein concentration of 18 mg / mL to 22 mg / mL. The pharmaceutical composition according to any one of claims 1 to 9.

11. The pharmaceutical composition comprises: (a) the antibody-drug conjugate having a protein concentration of 1 mg / mL to 100 mg / mL; (b) 0.1 mg / mL to 0.8 mg / mL of a poloxamer or polysorbate; (c) 30 mg / mL to 50 mg / mL sucrose; (d) 6 mg / mL to 15 mg / mL glycine, and (e) 10 mM to 50 mM succinic acid-sodium succinate buffer; the pH of the pharmaceutical composition is 3.5 to 5.5; Preferably, the pharmaceutical composition comprises: (a) the antibody-drug conjugate having a protein concentration of 1 mg / mL to 50 mg / mL; (b) 0.4 mg / mL to 0.8 mg / mL of a poloxamer or polysorbate; (c) 30 mg / mL to 50 mg / mL sucrose; (d) 6 mg / mL to 15 mg / mL glycine, and (e) 20 mM to 40 mM succinic acid-sodium succinate buffer; the pH of the pharmaceutical composition is 3.7 to 4.7; More preferably, the pharmaceutical composition comprises: (a) the antibody-drug conjugate at a protein concentration of 20 mg / mL; (b) 0.6 mg / mL poloxamer 188 or 0.2 mg / mL polysorbate 80; (c) 40 mg / mL sucrose; (d) 9 mg / mL glycine, and (e) 30 mM succinic acid-sodium succinate buffer; The pH of the pharmaceutical composition is 4.

2. The pharmaceutical composition according to any one of claims 1 to 10.

12. A lyophilized formulation comprising an antibody-drug conjugate, which, after reconstitution, can form the pharmaceutical composition of any one of claims 1 to 11.

13. A lyophilized formulation comprising an antibody-drug conjugate, the lyophilized formulation being obtainable by lyophilizing the pharmaceutical composition according to any one of claims 1 to 11.

14. A reconstitution solution containing an antibody-drug conjugate, the reconstitution solution being prepared by reconstituting the lyophilized formulation of claim 12 or 13.

15. A product comprising a container containing the pharmaceutical composition according to any one of claims 1 to 11, the lyophilized formulation according to claim 12 or 13, or the reconstitution solution according to claim 14.

16. 19. Use of the pharmaceutical composition according to any one of claims 1 to 11, the lyophilized formulation according to claim 12 or 13, the reconstituted solution according to claim 14 or the product according to claim 15 in the preparation of a medicament for treating a viral infection, a tumor or a cancer, wherein preferably the tumor is a cancer associated with expression of domain II of HER2, preferably the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric 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.

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