Pharmaceutical composition containing an anti-Nectin-4 antibody-drug conjugate and its use

Abstract

Provided are a pharmaceutical composition comprising an anti-Nectin-4 antibody-drug conjugate and its use. The provided pharmaceutical composition has good stability.

Description

Technical Field

[0001] This application claims priority to Chinese Patent Application CN202210530522.7, filed on May 16, 2022.

[0002] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing an anti-Nectin-4 antibody-drug conjugate, and its use as an anti-cancer agent.

Background Art

[0003] The descriptions herein do not necessarily constitute prior art, but only provide background information related to the present disclosure.

[0004] Nectin-4 (PVRL4) is a 66 kDa type I transmembrane glycoprotein that belongs to the Nectin family of the Ig superfamily and plays an important role in various biological processes (proliferation, differentiation, and migration) of epithelial cells, endothelial cells, immune cells, and nerve cells. Studies have shown that Nectin-4 plays an important role in the occurrence, invasion, and metastasis processes in malignant tumor tissues such as bladder cancer, breast cancer, and lung cancer. This role is related to the activation of Cdc42 and Rac and the change of cytoskeletal actin, and further causes the proliferation, invasion, and metastasis of tumor cells.

[0005] Nectin-4 is a tumor-specific expression antigen, and its expression rates in bladder cancer, breast cancer, and lung cancer reach 50%, 49%, and 86% respectively, and it is frequently found in tumors with poor prognosis, but its expression was not detected in most normal tissues. From these information, it is suggested that Nectin-4 can be an ideal target for tumor treatment.

[0006] Enfortumab vedotin (Padcev) is a conjugate that targets Nectin-4 and binds to MMAE. Studying more antibody-drug conjugates targeting Nectin-4 as anti-tumor drugs has important significance.

[0007] Since ADCs have a more complex heterostructure than antibodies, they pose even greater challenges for therapeutic ADC formulations.

SUMMARY OF THE INVENTION

[0008] The present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has a structure represented by the formula NEC49-9-A:

CHEMICAL

[0009] The present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has a structure represented by the formula NEC49-9-A:

CHEMICAL

[0010] In an alternative embodiment, the anti - Nectin - 4 antibody NEC49 described in the pharmaceutical composition comprises a heavy chain shown in SEQ ID NO: 6 (SEQ ID NO: 21 in PCT / CN2022 / 089129) and a light chain shown in SEQ ID NO: 7 (SEQ ID NO: 22 in PCT / CN2022 / 089129). TIFF2025521100000003.tif85155

[0011] TIFF2025521100000004.tif48155

[0012] In an alternative embodiment, the pH value in the pharmaceutical composition is from about 5.0 to about 6.5, non - limiting examples include about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, and any range between these point values, preferably a pH of about 5.5 to about 6.5, more preferably a pH of about 5.9 to about 6.2.

[0013] In an alternative embodiment, the concentration of the buffer in the pharmaceutical composition is from about 5 mM to about 50 mM, non - limiting examples include 5 mM, 10 mM, 12 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 40 mM, 45 mM, 50 mM and any range between these point values, preferably from about 10 mM to about 50 mM, more preferably from about 20 mM to about 40 mM, and most preferably about 30 mM.

[0014] In alternative embodiments, the pharmaceutical composition further comprises a surfactant. It may be selected from polysorbate, polysorbate 20, polysorbate 80, poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleoyl taurine sodium, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0015] In alternative embodiments, the concentration of the surfactant in the pharmaceutical composition is from about 0.01 mg / mL to about 1.0 mg / mL, preferably from about 0.05 mg / mL to about 0.6 mg / mL, more preferably from about 0.05 mg / mL to about 0.4 mg / mL, even more preferably from about 0.1 mg / mL to about 0.3 mg / mL, or from about 0.2 mg / mL to about 0.6 mg / mL, most preferably about 0.2 mg / mL. Non-limiting examples include about 0.01 mg / mL, 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, 0.9 mg / mL, 1.0 mg / mL, and any range between these point values.

[0016] In alternative embodiments, the pharmaceutical composition further comprises a sugar. The "sugar" of the present disclosure refers to ordinary compositions (CH2O) n and its derivatives, 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. Preferred sugars are non-reducing disaccharides, more preferably selected from α,α-trehalose dihydrate or sucrose, and most preferably sucrose.

[0017] In alternative embodiments, the concentration of the sugar in the pharmaceutical composition is about 25 mg / mL to about 80 mg / mL, preferably about 30 mg / mL to about 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, preferably 40 mg / mL.

[0018] In alternative embodiments, the pharmaceutical composition further comprises an amino acid, which is preferably glycine.

[0019] In a selective embodiment, the concentration of the amino acid in the pharmaceutical composition is about 6 mg / mL to about 15 mg / mL, about 7 mg / mL to about 11 mg / mL, preferably about 7 mg / mL to about 10 mg / mL, more preferably about 7 mg / mL to about 9 mg / mL, about 8 mg / mL to about 9 mg / mL, and non-limiting examples include about 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, 9.5 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, and most preferably about 9 mg / mL.

[0020] In a selective embodiment, the concentration of glycine in the pharmaceutical composition is about 6 mg / mL to about 15 mg / mL, about 7 mg / mL to about 11 mg / mL, preferably about 7 mg / mL to about 10 mg / mL, more preferably about 7 mg / mL to about 9 mg / mL, about 8 mg / mL to about 9 mg / mL, and non-limiting examples include about 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, 9.5 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, and most preferably about 9 mg / mL.

[0021] In alternative embodiments, the concentration of the antibody-drug conjugate in the pharmaceutical composition is a protein concentration of from about 1 mg / mL to about 100 mg / mL, non-limiting examples of which 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, 24 mg / 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. Preferably, the concentration of the antibody-drug conjugate is a protein concentration of from about 10 mg / mL to about 30 mg / mL, more preferably, the concentration of the antibody-drug conjugate is a protein concentration of from about 18 mg / mL to about 22 mg / mL, and most preferably about 20 mg / mL. Specifically, 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, 20.9 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 "in terms of protein concentration" means measured in terms of the concentration of the antibody moiety in the antibody-drug conjugate.

[0022] In alternative embodiments, the range of the drug loading (n) may be the average number of cytotoxic drugs bound to each anti-Nectin-4 antibody. As a non-limiting example, the average number of cytotoxic drugs bound to each antibody may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and any range between these point values. Preferably, it is selected from 1-10, 1-8, 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. Exemplarily, the drug loading (n) may be the average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. n may be a decimal or an integer.

[0023] In alternative embodiments, the drug loading (n) is about 3.5-4.7.

[0024] In alternative embodiments, the drug loading (n) is about 4.

[0025] In alternative embodiments, the above pharmaceutical composition, which (a) the above antibody-drug conjugate at a protein concentration of about 1 mg / mL to about 100 mg / mL, (b) about 0.05 mg / mL to about 0.4 mg / mL of polysorbate, (c) about 30 mg / mL to about 50 mg / mL of sucrose or α,α-trehalose dihydrate, (d) about 6 mg / mL to about 15 mg / mL of glycine, and (e) about 10 mM to about 50 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.5-6.5.

[0026] In alternative embodiments, the above pharmaceutical composition, which (a) the above antibody-drug conjugate at a protein concentration of about 10 mg / mL to about 30 mg / mL, (b) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate, (c) about 30 mg / mL to about 50 mg / mL of sucrose or α,α-trehalose dihydrate, (d) about 7 mg / mL to about 11 mg / mL of glycine, and (e) about 10 mM to about 50 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.5-6.5.

[0027] In a selected embodiment, the above pharmaceutical composition, which (a) the above antibody-drug conjugate at a protein concentration of about 10 mg / mL to about 30 mg / mL, (b) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 80, (c) about 30 mg / mL to about 50 mg / mL of sucrose, (d) about 7 mg / mL to 11 mg / mL of glycine, and (e) about 10 mM to about 50 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.5 to 6.5.

[0028] In a selected embodiment, the above pharmaceutical composition, which (a) the above antibody-drug conjugate at a protein concentration of about 10 mg / mL to about 30 mg / mL, (b) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 80, (c) about 30 mg / mL to about 50 mg / mL of sucrose, (d) about 7 mg / mL to 11 mg / mL of glycine, and (e) about 20 mM to about 40 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.5 to 6.5.

[0029] In a selected embodiment, the above pharmaceutical composition, which (a) the above antibody-drug conjugate at a protein concentration of about 18 mg / mL to about 22 mg / mL, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 9 mg / mL of glycine, and (e) about 30 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.9 to 6.2.

[0030] In a selected embodiment, the above pharmaceutical composition, which (a) the above antibody-drug conjugate at a protein concentration of about 20 mg / mL, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 9 mg / mL of glycine, and (e) about 30 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 6.0.

[0031] In alternative embodiments, any one of the above pharmaceutical compositions is a liquid formulation. The liquid formulations or reconstituted formulations of the present disclosure have relatively good stability. Further, stable liquid formulations include those that exhibit desired properties when stored at a temperature including 40°C for one month.

[0032] The present disclosure further provides a lyophilized formulation comprising an antibody-drug conjugate, characterized in that the above pharmaceutical composition can be formed after reconstituting the formulation.

[0033] 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 above pharmaceutical composition.

[0034] In alternative embodiments, among the methods for preparing a lyophilized formulation comprising an antibody-drug conjugate, the lyophilization includes the steps of pre-freezing, primary drying, and secondary drying in sequence. Lyophilization is performed by freezing the formulation and subsequent sublimation of water at a temperature suitable for primary drying. Under these conditions, the temperature of the product is lower than the eutectic point or collapse temperature of the formulation. Usually, the temperature range for primary drying is about -30°C to 25°C (assuming the product remains frozen during the primary drying process). The time required for drying is determined by the formulation, the size and type of the container (e.g., glass vial) containing the sample, and the volume of the liquid, and the range of the time may be from several hours to several days (e.g., 40 hours to 60 hours). The secondary drying stage may be carried out at about 0°C to 40°C, which is mainly determined by the type and size of the container and the type of protein used. The time for secondary drying is determined by the desired remaining moisture level in the product and usually at least about 5 hours is required. Usually, the water content of the lyophilized formulation is lower than about 5%, preferably lower than about 3%. The pressure may be the same as the pressure applied in the primary drying step, and preferably, the pressure for secondary drying is lower than that for primary drying. The lyophilization conditions may be varied depending on the formulation and the size of the vial.

[0035] In one alternative embodiment of the present disclosure, 4.4 mL of the stock composition is lyophilized, and the lyophilization procedure is as follows: the pre-freezing temperatures are 5°C and -45°C in sequence, the temperature for primary drying is -20°C, the vacuum degree is 10 Pa, the temperature for secondary drying is 25°C, and the vacuum degrees are 10 Pa and 1 Pa in sequence.

[0036] 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.

[0037] The present disclosure further provides a lyophilized formulation comprising an antibody-drug conjugate, which is obtained by lyophilizing the pharmaceutical composition of the above antibody-drug conjugate.

[0038] The present disclosure further provides a reconstitution solution comprising an antibody-drug conjugate, which is characterized by being prepared by reconstituting the above lyophilized formulation.

[0039] The present disclosure further provides a method for preparing the above reconstitution solution, which includes the step of reconstituting the above lyophilized formulation, and the solution used for reconstitution is selected from, but not limited to, water for injection, physiological saline, or glucose solution.

[0040] In an alternative embodiment, the above reconstitution solution contains (a) the above antibody-drug conjugate at a protein concentration of about 1 mg / mL to about 100 mg / mL, (b) polysorbate at about 0.05 mg / mL to about 0.4 mg / mL, (c) sucrose or α,α-trehalose dihydrate at about 30 mg / mL to about 50 mg / mL, (d) glycine at about 6 mg / mL to about 15 mg / mL, and (e) a histidine-hydrochloric acid histidine buffer at about 10 mM to about 50 mM, and the pH of the above pharmaceutical composition is about 5.5 to 6.5.

[0041] In an alternative embodiment, the above reconstitution solution (a) At a protein concentration of about 10 mg / mL to about 30 mg / mL of the above antibody-drug conjugate, (b) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate, (c) about 30 mg / mL to about 50 mg / mL of sucrose or α,α-trehalose dihydrate, (d) about 7 mg / mL to about 11 mg / mL of glycine, and (e) about 10 mM to about 50 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.5 to 6.5.

[0042] In alternative embodiments, the above reconstitution solution (a) At a protein concentration of about 10 mg / mL to about 30 mg / mL of the above antibody-drug conjugate, (b) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 80, (c) about 30 mg / mL to about 50 mg / mL of sucrose, (d) about 7 mg / mL to about 11 mg / mL of glycine, and (e) about 10 mM to about 50 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.5 to 6.5.

[0043] In alternative embodiments, the above reconstitution solution (a) At a protein concentration of about 10 mg / mL to about 30 mg / mL of the above antibody-drug conjugate, (b) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 80, (c) about 30 mg / mL to about 50 mg / mL of sucrose, (d) about 7 mg / mL to about 11 mg / mL of glycine, and (e) about 20 mM to about 40 mM of histidine-hydrochloride histidine buffer, and the pH of the above pharmaceutical composition is about 5.5 to 6.5.

[0044] In alternative embodiments, the above reconstitution solution (a) About 18 mg / mL to about 22 mg / mL of the above antibody-drug conjugate, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 9 mg / mL of glycine, and (e) about 30 mM of histidine-hydrochloride histidine, and the pH of the above pharmaceutical composition is about 5.9 to 6.2.

[0045] In alternative embodiments, the above reconstitution solution (a) The above antibody-drug conjugate at about 20 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 9 mg / mL, and (e) histidine-hydrochloride histidine at about 30 mM, and the pH of the above pharmaceutical composition is about 6.0.

[0046] The present disclosure further provides a kit comprising a container containing the above pharmaceutical composition, lyophilized formulation or reconstitution solution. In some embodiments, the container is an injection bottle made of neutral borosilicate glass tubing.

[0047] The present disclosure further provides the use of the above pharmaceutical composition or lyophilized formulation or reconstitution solution in the preparation of a medicament for treating or preventing tumors.

[0048] The present disclosure further provides a method of treating a disease, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the above pharmaceutical composition or lyophilized formulation or reconstitution solution or kit.

[0049] The present disclosure further provides the above pharmaceutical composition, or lyophilized formulation, or reconstitution solution, or kit as a drug for use in the treatment or prevention of diseases.

[0050] The present disclosure further provides the above pharmaceutical composition, or lyophilized formulation, or reconstitution solution, or kit as a drug for use in the treatment or prevention of tumors.

[0051] In alternative embodiments, the above disease or tumor is a Nectin-4 mediated disease or condition.

[0052] The present disclosure further provides the use of the above pharmaceutical composition as a medicament, or a lyophilized preparation, or a reconstituted solution, or a kit in the preparation of a medicament for treating viral infection, tumor or cancer, wherein the tumor or cancer is urothelial cancer (e.g., metastatic urothelial cancer, locally advanced urothelial cancer), ureteral cancer, breast cancer, pancreatic cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), esophageal cancer (also referred to as "esophageal cancer"), laryngeal tumor, pharyngeal tumor, oral tumor, gastric cancer, ovarian cancer, prostate cancer (e.g., metastatic castration-resistant prostate cancer), bladder cancer (locally advanced bladder cancer), colorectal cancer (including colon cancer and rectal cancer), head and neck cancer, squamous cell carcinoma and melanoma.

[0053] In some embodiments, it is the pharmaceutical composition, or the lyophilized preparation, or the reconstituted solution, or the kit according to any one of the above, which is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation or a intramuscular injection preparation. In some embodiments, it is an intravenous injection preparation.

[0054] As is well known to those skilled in the art, one, several or all of the characteristics of each embodiment described in the present disclosure can further be combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments obtained by combination will be further described by the following detailed description.

Mode for Carrying Out the Invention

[0055] The present disclosure provides a pharmaceutical composition that is advantageous in manufacture and administration and has stable performance. Among them, undesirable instabilities 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, dissociation of toxins, etc. Specifically, the pharmaceutical composition described in the present disclosure includes an antibody-drug conjugate and a buffer.

[0056] Term To make the present disclosure more easily understood, several technical and scientific terms are specifically defined below. Unless otherwise specifically defined in the text, all other technical and scientific terms used in the text have meanings generally understood by those skilled in the art.

[0057] "Antibody-drug conjugate (ADC)" is a complex formed by linking an antibody (or its antigen-binding fragment) to a cytotoxic agent having biological activity or a small molecule drug having cell-killing activity via a linker unit.

[0058] "Drug loading amount" or "drug payload" is also referred to as the drug-to-antibody ratio (DAR), that is, the average number of drugs coupled to each antibody in the ADC. It may be, for example, within the range where each antibody is coupled to about 1 to about 10 drugs, and in certain embodiments, within the range where each antibody is coupled to about 1 to about 8 drugs, preferably selected from integers or decimals of 1-8, 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 3-6, 4-5, 5-6, 5-7, 5-8, 6-7, and 6-8. Exemplarily, the drug loading amount may be an average value obtained from any of the numerical values of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The general formula of the ADC according to the present disclosure includes the set of antibodies coupled to drugs within the above certain range. In embodiments of the present disclosure, the drug loading amount may be indicated by n. The drug loading amount can be measured by ordinary methods such as UV / visible spectroscopy, mass spectrometry, ELISA test, and HPLC.

[0059] The term "linker unit" or "linking fragment" or "linking unit" refers to a chemical structural fragment or bond having one end linked to an antibody or its antigen-binding fragment and the other end linked to a drug, and may be linked to the drug after being linked to another linker.

[0060] The linker includes an extender, a spacer, and amino acid units, and can 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 photo-labile 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.

[0061] The term "drug-linker fragment" or "drug-linker moiety" refers to a fragment in which a drug is linked to a linker unit, which can be linked to an antibody or an antigen-binding fragment thereof via the other end of the linker unit.

[0062] The loading amount of the cytotoxic drug (1) controlling the molar ratio of the conjugated drug-linker fragment to the antibody, (2) controlling the reaction time and temperature, (3) selecting different reaction reagents, and can be controlled by non-limiting methods including these.

[0063] The three-letter and one-letter codes of the amino acids used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0064] The term "antibody" as used in this disclosure is used in the broadest sense if it exhibits the desired antigen-binding activity and covers various antibody structures, including monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), but is not limited thereto.

[0065] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As one of the more preferred prior arts, the mammalian expression system will cause glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. The positive clones are expanded in a serum-free medium of a bioreactor to produce the antibody. The culture broth in which the antibody is secreted can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing a conditioned buffer. The nonspecifically bound components are washed away. Further, the bound antibody is eluted by a pH gradient method, and the antibody fragment is detected and collected by SDS-PAGE. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers may be removed by conventional methods such as molecular sieving and ion exchange. The resulting product must be immediately frozen at -70°C or freeze-dried.

[0066] "Buffer" refers to a buffer that withstands 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.

[0067] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include buffers such as histidine - histidine hydrochloride, histidine - histidine acetate, histidine - histidine phosphate, histidine - histidine sulfate, etc., preferably a histidine - histidine hydrochloride buffer. The histidine - histidine hydrochloride buffer can be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0068] "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.

[0069] "Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid - sodium succinate salt, succinic acid - potassium succinate, succinic acid - calcium succinate salt, etc. A preferred succinate buffer is succinic acid - sodium succinate salt. Exemplarily, the above-mentioned succinic acid - sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate salt.

[0070] "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.

[0071] "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.

[0072] "Pharmaceutical composition" represents 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 vectors and excipients. The pharmaceutical composition is for maintaining the stability of the active ingredient of the antibody, promoting administration to the living body, contributing to the absorption of the active ingredient, and further exerting biological activity.

[0073] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0074] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0075] "Lyophilized formulation" refers to a formulation or pharmaceutical composition obtained after performing a vacuum freeze-drying step on a pharmaceutical composition or liquid or solution formulation in liquid or solution form.

[0076] As used herein, the terms "about" and "substantially" mean that the numerical value is within the acceptable error range of the specific value measured by those skilled in the art, depending on how the above numerical part is measured or measured (i.e., the limitations of the measurement system). For example, in each execution in this field, "about" may mean within 1 or the standard deviation exceeding 1. Alternatively, "about" or "substantially includes" may mean a range of at most 20%. Furthermore, especially for biological systems or processes, the term may mean at most one order of magnitude or at most 5 times the numerical value. Unless otherwise specified, when a specific value appears in the present application and the claims, the meaning of "about" or "substantially includes" should be assumed to be within the acceptable error range of the specific value.

[0077] The pharmaceutical composition described in the present disclosure can achieve a stable effect, that is, after the antibody-drug conjugate therein is stored, it basically retains its physical stability and / or chemical stability and / or biological activity. Preferably, after the pharmaceutical composition is stored, it basically retains its physical and chemical stability and its biological activity. The storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition. At present, there are multiple analytical techniques for measuring the stability of proteins or antibody-drug conjugates, and the stability after storage at a predetermined temperature for a predetermined period can be measured.

[0078] A stable formulation is a formulation that shows no significant change when stored at refrigeration temperature (2°C to 8°C) for at least 3 months, preferably 6 months, more preferably 1 year. Also, a stable liquid formulation includes a liquid formulation that exhibits the desired characteristics after being stored for a period including 1 month, 2 months, 3 months at a temperature including 25°C. Further, a stable liquid formulation includes a liquid formulation that exhibits the desired characteristics after being stored for a period including 10 days, 20 days, 1 month at a temperature including 40°C. As a typical example of stability, it is measured by SEC-HPLC that the antibody monomer that has aggregated or decomposed usually does not exceed about 10%, preferably does not exceed about 5%. By visual analysis, the formulation is a pale yellow, almost colorless and transparent liquid, or colorless, or clear to slightly milky white. The concentration, pH and osmolarity of the above formulation have a change not exceeding ±10%. Usually, a decrease not exceeding about 10%, preferably not exceeding about 5% is observed. Usually, aggregation not exceeding about 10%, preferably not exceeding about 5% is formed.

[0079] When the color and / or clarity are inspected visually, or measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS), if the antibody-drug conjugate shows no significant increase in aggregation, precipitation and / or denaturation, the antibody-drug conjugate is considered to "maintain its physical stability" in the pharmaceutical formulation. The change in the conformation of the protein can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0080] If the antibody-drug conjugate shows no significant chemical changes, the antibody-drug conjugate is considered to "maintain its chemical stability" in the pharmaceutical formulation. Chemical stability can be evaluated by detecting and quantifying the chemically modified form of the protein. The degradation processes that constantly change the chemical structure of the protein include hydrolysis or cleavage (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as mass spectrometry or peptide mapping combined with MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing electrophoresis, peptide mapping, and measurement of isoaspartic acid), and isomerization (evaluated by measurement of the content of isoaspartic acid, peptide mapping, etc.).

[0081] If the biological activity of the antibody-drug conjugate is within the predetermined range of the biological activity shown when preparing the pharmaceutical formulation at a given time, the antibody-drug conjugate is considered to "maintain its biological activity" in the pharmaceutical formulation.

[0082] "Optional" or "optionally" means that the event or situation described thereafter may or may not occur, and this description includes both the case where the event or situation occurs and the case where it does not occur. For example, "optionally containing 1 to 3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may or may not be present.

[0083] The preparation of ordinary pharmaceutical compositions is shown in the Chinese Pharmacopoeia.

[0084] As used in the agents of the present disclosure, the term "vector" refers to a system that can change the way a drug enters the human body and its distribution in the body, control the release rate of the drug, and transport the drug to the target organ. The release of the drug vector and the target system can reduce the decomposition and loss of the drug, reduce side effects, and improve bioavailability. For example, polymeric surfactants used as vectors can self-assemble to form aggregates in various forms due to their unique amphiphilic structure. Preferred examples include, for example, micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, and can be used as good drug vectors.

[0085] "Administration" and "treatment", when applied to animals, humans, experimental subjects, cells, tissues, organs, and biological fluids, mean the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" may, for example, mean therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of the reagent with the cells and contact of the reagent with a fluid, where the fluid contacts the cells. "Administration" and "treatment" also mean treating cells, for example, in vitro and ex vivo, with a reagent, diagnostic, binding composition, or through another type of cell. "Treatment" means therapeutic treatment, prophylaxis or preventive measures, research, and diagnostic use when applied to humans, veterinary medicine, or research subjects.

[0086] "Treatment" means administering to a patient an orally or topically administered therapeutic agent, e.g., a composition comprising any one of the antibody-drug conjugates of the present disclosure, where the patient has one or more disease symptoms and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the patient or population being treated is given the therapeutic agent in an amount effective to alleviate one or more disease symptoms, thereby inducing regression of these symptoms or inhibiting the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as the "therapeutically effective amount") can vary depending on various factors such as the patient's disease state, age and weight, and the ability of the agent to produce the required therapeutic effect in the patient. The reduction of disease symptoms can be evaluated by any clinically detectable method commonly used by a physician or other professional healthcare provider to assess the severity or progression of the symptoms. Embodiments of the present disclosure (e.g., treatment methods or kits) may be ineffective in alleviating each respective target disease symptom, but are confirmed to reduce the target disease symptoms in a statistically significant number of patients by any statistical testing method known in the art, such as the Student t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0087] "Effective amount" includes an amount sufficient for the amelioration or prevention of the symptoms or conditions of a medical disease. Effective amount further refers to an amount sufficient for the allowance or facilitation of a diagnosis. The effective amount used for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method of administration, route and dosage, and the severity of side effects. The effective amount may be the maximum dosage or dosing schedule at which significant side or toxic effects are avoided.

[0088] "Replacement" refers to the replacement of the solvent system that dissolves the antibody protein or antibody-drug conjugate. For example, the high-salt or high-osmotic-pressure solvent system containing the antibody protein or antibody-drug conjugate is replaced by a physical operation method using the buffer system of the stable formulation so that the antibody protein or antibody-drug conjugate is present in the stable formulation. The above physical operation methods include, but are not limited to, ultrafiltration, dialysis, or redissolution after centrifugation.

Example

[0089] Hereinafter, the present disclosure will be further described in conjunction with examples, but these examples do not 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 normal conditions such as those published by Cold Spring Harbor Laboratory in "Antibody Technology Experimental Manual" and "Molecular Cloning Manual", or follow the conditions recommended by the raw material or product manufacturer. Reagents for which specific sources are not specified are ordinary commercially available reagents.

[0090] I. Preparation of Antibody Example 1. Construction of Nectin-4 High-Expression Cell Line The PBABE-Nectin4 lentiviral expression vector plasmid and the pVSV-G and pGag-pol lentiviral packaging vectors were transfected into the viral packaging cell 293T with Lipofectamine 3000 transfection reagent. The culture supernatant containing the virus was collected, filtered, centrifuged at ultra-high speed, and concentrated virus was used to infect Chinese hamster ovary cells CHO-K1. After screening with puromycin for 2 to 3 weeks, FACS single-cell sorting was performed.

[0091] The expression of Nectin-4 on the surface of lentivirus-infected CHO-K1 cells was detected by FACS, and monoclonal cell lines with high Nectin-4 expression levels were selected, expanded in culture, and prepared for cryopreservation.

[0092] TIFF2025521100000005.tif85160

[0093] The positive control antibody EV201 was prepared with reference to (WO2012047724, page 115). Among them, the amino acid sequences of the heavy and light chains of EV201 (enfortumab vedotin) are as follows.

[0094] TIFF2025521100000006.tif80160

[0095] TIFF2025521100000007.tif48155

[0096] Example 2. Screening of anti-human Nectin-4 monoclonal antibodies The antibodies of the present disclosure are cited from NEC49 in PCT / CN2022 / 089129. PCT / CN2022 / 089129 is incorporated herein by reference in its entirety. Using a fully human semi-synthetic phage antibody library and the antigen Biotinylated Human Nectin-4 (purchased from Beijing ACROBiosystems Co., Ltd., product number: NE4-H82E7), after screening, phages were detected by the ELISA method to obtain positive clones. After sequencing the positive clones to obtain the sequences, the positive clone sequences were inserted into the protein expression vector Phr-IgG and expressed in HEK293 and Expi-CHO-S. After purification, FACS and endocytosis activity verification experiments were performed, and finally the fully human antibody molecule NEC49 was selected.

[0097] The variable region sequences of the fully human antibody molecule NEC49 are as follows.

[0098] TIFF2025521100000008.tif38155

[0099] TIFF2025521100000009.tif32155

[0100] [Table 1]

[0101] The above anti-Nectin4 antibody may further include an antibody heavy chain constant region and a light chain constant region. The above heavy chain constant region may be selected from human IgG1, IgG2, IgG3, and IgG4 constant regions, and the above light chain constant region may be selected from human antibody κ and λ chain constant regions. In the present disclosure, the above antibody includes a heavy chain constant region of human IgG1 and a light chain constant region of human κ. The sequence of the antibody molecule NEC49 of the present disclosure is as follows.

[0102] TIFF2025521100000011.tif85155

[0103] TIFF2025521100000012.tif48155

[0104] II. Construction of ADC Molecule Measurement of ADC Drug Loading The drug loading of the ADC was measured by reverse-phase high-performance liquid chromatography (RP-HPLC).

[0105] 1. Measurement method: 4 μL of DDT (sigma) was added to the naked antibody and the sample ADC to be measured (concentration 1 mg / mL) for reduction, and they were incubated in a water bath at 37°C for 1 hour. After completion, they were taken out and placed in an inner tube. Detection was performed using a high-performance liquid chromatograph Agilent 1200. Agilent PLRP-S 1000A 8 μm 4.6×250 mm was selected as the column, the column temperature was 80°C, the wavelength of the DAD detector was 280 nm, the flow rate was 1 mL / min, and the injection volume was 40 μL. Then, by comparing the spectra of the sample and the naked antibody, the positions of the light chain and heavy chain were distinguished, and integration was performed on the spectrum of the detection sample to calculate the DAR value.

[0106] 2. Solution preparation 1) 0.25 M DTT solution: 2) Mobile phase A (0.1% aqueous TFA solution): 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0107] 3. Data Analysis By comparing the spectra of the sample and the naked antibody, the positions of the light chain and heavy chain were distinguished, and then the spectrum of the detection sample was integrated to calculate the drug loading DAR value. The calculation formula is as follows.

[0108]

Table 2

[0109] Sum of LC peak areas = LC peak area + LC +1 peak area Sum of HC peak areas = HC peak area + HC +1 peak area + HC +2 peak area + HC +3 peak area LC DAR = Σ(Number of conjugated drugs × Peak area percentage) / Sum of LC peak areas HC DAR = Σ(Number of conjugated drugs × Peak area percentage) / Sum of HC peak areas DAR = LC DAR + HC DAR.

[0110] Example 3-1. ADC-1

Chemical formula

[0111] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (10 mM, 130 μL, 1300 nmol) prepared was added to the PBS buffer aqueous solution of antibody NEC49 (0.05 M PBS buffer aqueous solution with pH = 6.5, 10.0 mg / mL, 6.4 mL, 432 nmol), placed in a water bath shaker, and reacted while shaking at 37 °C for 3 hours, and the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0112] Compound 9-A (prepared with reference to 9-A in Example 9 on pages 58 - 60 of WO2020063676, 5.6 mg, 5214 nmol) was dissolved in 300 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution (2.89 mg / mL, 21 mL) of the exemplary product ADC-1 of the title complex general formula NEC49-9-A was obtained and stored refrigerated at 4 °C. The average value of the loading amount was calculated by RP-HPLC: n = 4.90.

[0113] Example 3-2. ADC-2

Chemical formula

[0114] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (10 mM, 28.5 μL, 285 nmol) was added to an aqueous PBS buffer solution of the control antibody EV201 (0.05 M PBS buffer aqueous solution with pH = 6.5, 10.0 mg / mL, 1.63 mL, 110 nmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0115] Compound 9-A (1.18 mg, 1100 nmol) was dissolved in 90 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA), and a PBS buffer solution (1.12 mg / mL, 12 mL) of the exemplary product ADC-2 of the title complex general formula EV201-9-A was obtained and stored refrigerated at 4 °C. The average value of the loading amount was calculated by RP-HPLC: n = 3.90.

[0116] Example 3-3. ADC-3 [Chemical formula]

[0117] Under the condition of 37 °C, an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (10 mM, 64.4 μL, 644 nmol) prepared was added to an aqueous PBS buffer solution of antibody EV201 (0.05 M PBS buffer solution at pH = 6.5, 10.0 mg / mL, 3.67 mL, 248 nmol), placed in a water bath shaker, and reacted with shaking at 37 °C for 3 hours, and then the reaction was stopped. The reaction solution was cooled to 25 °C in a water bath.

[0118] Compound VcMMAE (HanXiang Biotechnology, CAS 646502-53-6, 3.3 mg, 2480 nmol) was dissolved in 150 μL of DMSO, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25 °C for 3 hours, and then the reaction was stopped. The reaction solution was desalted and purified by a Sephadex G25 gel column (eluent: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA), and an example product ADC-3 of the title conjugate, a PBS buffer solution of general formula EV201-MMAE (2.71 mg / mL, 14 mL) was obtained and stored refrigerated at 4 °C. The average value of the loading amount was calculated by RP-HPLC: n = 4.09.

[0119] Example 3-4. ADC-4 [Chemical formula]

[0120] Under the condition of 12 °C, in a 20 mM histidine-acetic acid-Tris buffer solution (pH 7.3) containing 2.5 mM EDTA, 186.37 g of antibody NEC49 (1.28 mmol, diluted to an antibody concentration of 23 mg / mL with 20 mM histidine-acetic acid) was reacted with 0.8873 g of tris(2-carboxyethyl)phosphine hydrochloride (Sigma, 3.10 mmol) with stirring in a constant temperature water bath for 100 minutes to generate an intermediate I solution. 2 M acetic acid was added dropwise to adjust the pH of the reaction solution to 5.0.

[0121] Compound 9-A (6.13 g, 5.71 mmol) was dissolved in 0.446 L of DMSO to form a DMSO solution of Compound 9-A. After adding 0.365 L of DMSO to the above intermediate I solution in advance, the DMSO solution of Compound 9-A was added to the intermediate I solution with DMSO added in advance, and the reaction was carried out with stirring at 12 °C for 100 minutes, and the reaction was stopped. Product ADC-4 was obtained (20 g / L, 175.93 g, average value calculated by reverse-phase chromatography: n = 4.0).

[0122] Biological tests Test Example 1. Affinity and kinetic experiments of antibodies at the protein level The anti-Nectin-4 antibody was analyzed by Biacore T200 (GE) to characterize its affinity and binding kinetics. The IgG antibody was affinity captured with a Protein A biosensor chip (Cat.#29127556, GE), and then a series of concentration gradients of human Nectin-4 His (Cat.#19771-H08H, Sino Biological) antigen diluted in HBS-EP buffer (pH 7.4) (Cat.#BR-1001-88, GE) was flowed over the chip surface. The antigen-antibody binding kinetics was traced for 3 minutes, and the dissociation kinetics was traced for 10 minutes. The reaction signal was detected in real time by the Biacore T200 instrument to obtain the binding and dissociation curves. After the dissociation of each experimental cycle was completed, the biosensor chip was washed and regenerated with 10 mM Gly-HCl pH 1.5 (Cat.#BR-1003-54, GE). The obtained data was analyzed by GE's BIAevaluation software using a 1:1 (Langmuir) binding model, and the ka (kon), kd (koff), and KD values measured by this method are shown in Table 2-1.

[0123] [Table 3]

[0124] Test Example 2. In vitro cell binding experiment of antibodies Nectin4-CHOK1 / T47D / MDA-MB-468 cells were adjusted to a cell suspension of 1×10 6 / mL with FACS buffer (2% fetal bovine serum (Gibco, 10099141), PBS (Sigma, P4417-100TAB) at pH 7.4), and 100 μL / well was added to a 96-well round-bottom plate. After centrifugation to remove the supernatant, various concentrations of the antibody to be measured diluted with FACS buffer were added at 50 μL / well, and the plate was placed in a refrigerator at 4°C and incubated in the dark for 1 hour. After washing by centrifugation three times at 500 g with FACS buffer, Alexa Fluor 488 Goat anti-Human IgG(H+L) (Invitrogen, A-11013) at the working concentration was added, and the plate was placed in a refrigerator at 4°C and incubated in the dark for 40 minutes. After washing by centrifugation three times at 500 g with FACS buffer, the geometric mean fluorescence intensity was detected with a BD FACSCantoII flow cytometer, and the binding EC 50 value of the antibody to Nectin-4-expressing cells was calculated. The results are shown in Table 2-2.

[0125]

Table 4

[0126] Conclusion: NEC49 has binding activity to multiple cell lines expressing Nectin-4 and is superior to the control molecule.

[0127] Test Example 3. Endocytosis Experiment of DT3C Antibody The purpose of this experiment is to indirectly reflect the endocytosis situation of the anti-Nectin-4 antibody based on the killing of activated DT on cells after the DT3C protein invades the cells. IC 50 and the maximum killing value were used to evaluate the in vitro endocytosis activity of the antibody.

[0128] DT3C is a recombinant fusion protein formed by the fusion of Fragment A of diphtheria toxin (only the toxin part) and the 3C fragment (IgG-binding part) of group G streptococcus. This protein can highly affinity bind to the Fc part of antibodies. When endocytosis occurs in the antibody, it enters the cell together. Under the action of intracellular furin, it releases the toxic DT. DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process, and ultimately cause cell death. DT3C that has not entered the cell does not have the activity to kill cells. The endocytosis activity of antibodies was evaluated according to the cell killing situation.

[0129] Prepare a Nectin4-CHOK1 cell suspension in fresh cell culture medium containing 20% low IgG FBS, with a cell density of 2×10 4 cells / mL. Add 50 μL / well to cell culture plate 3903 and culture at 5% carbon dioxide and 37 °C for 16 hours. Dilute DT3C to 1.6 μM in serum-free medium and dilute the antibody to 266.4 nM in serum-free medium. Mix 80 μL of DT3C and 80 μL of the antibody uniformly in a 1:1 volume ratio and incubate at room temperature for 30 minutes. The molar concentration of DT3C was 6 times that of the antibody molar concentration.

[0130] Dilute the DT3C and antibody mixture in a 4-fold gradient in serum-free medium with the 9th and 10th points being pure medium, a total of 8 gradients. C25-IgG1 is a homogenous IgG1 negative control group. Take 50 μL of the diluted mixture and add it to 50 μL of cells, and incubate in an incubator for 3 days. Add 50 μL of CTG to each well, incubate at room temperature in the dark for 10 minutes, attach a white bottom film to the bottom of the cell culture plate, and place it on a plate reader Victor3 to read the chemiluminescence value. The results are shown in Table 3.

[0131]

Table 5

[0132] Test Example 4. Endocytosis Experiment of pHrodo Antibody The purpose of this experiment is to reflect the endocytosis status of the Nectin-4 antibody by the change in the fluorescence signal after the dye is internalized. The in vitro endocytosis activity of the antibody was evaluated by the intensity of the fluorescence signal.

[0133] The Fab fragment coupled to the pH-sensitive pHrodo iFL dye can bind directly to the Fc region of the Nectin-4 antibody without affecting the antigen recognition of the antibody. The pHrodo iFL dye emits almost no fluorescence at neutral pH values. Simultaneously with the endocytosis of the Nectin-4 antibody, the dye is internalized, and as the pH decreases, the fluorescence signal gradually becomes stronger. The endocytosis activity of the antibody was evaluated by the enhancement status of the fluorescence signal.

[0134] Nectin4-CHOK1 clone3 cells were cultured in DMEM / F12 + 10% FBS + 10 μg / mL puromycin. On the first day of the experiment, a cell suspension with a density of 2×10 5 cells / mL was prepared with a fresh cell-containing medium and added to a 96-well cell culture plate at 100 μL / well, and cultured at 5% carbon dioxide and 37 °C for 24 hours.

[0135] Preparation of 4× antibody: The mother liquor of the antibody was diluted 10-fold with a medium without FBS to prepare a culture solution, and the culture solution was further diluted to 4× Dosing Solution (80 nM). The final concentration of the antibody was 20 nM.

[0136] 4× Zenon TM pHrodo TM Preparation of 4× pHrodo iFL IgG labeling reagent: The mother liquor of the labeling reagent was diluted to 4× concentration (240 nM) with a medium without FBS, and the final concentration was 60 nM. The above 4× antibody solution and 4× pHrodo TM labeling reagent solution TMThe labeling reagent solutions were mixed in equal volumes and incubated at room temperature for 10 minutes. 50 μL of the mixture was added to 150 μL of serum-free medium such that each antibody sample had two concentrations (20 nM and 5 nM).

[0137] 50 μL was aspirated from the cell lysate in a 384-well plate, 50 μL of the antibody and pHrodo dye mixture was added to each well, and two parallel wells were set up for each antibody sample. Also, a dye-alone group and a homotypic IgG1 control group were set up.

[0138] After culturing in an incubator for 24 hours, the medium was aspirated, 50 μL of pancreatin was added to each well, digestion was carried out for 2 minutes, and the digestion was stopped with 50 μL of fresh medium. The cells in the parallel wells of the same sample were transferred to the same well of a round-bottom plate using a multi-channel pipette. Centrifugation was carried out at 1500 rpm for 2 minutes, the culture solution was discarded, the cells were washed once with FACS buffer (PBS + 2.5% FBS), and centrifugation was carried out at 1500 rpm for 2 minutes. 200 μL of FACS buffer (PBS + 2.5% FBS) was added, the cells were resuspended, and the FITC signal was detected using a flow cytometer. The data was analyzed using Flowjo 7.6. The results of the endocytosis experiment in Nectin4-CHOK1 clone 3 of NEC49 and EV201 (C25-IgG1 was the homotypic IgG1 negative control group) are shown in Table 4.

[0139]

Table 6

[0140] NEC49 has endocytosis activity in Nectin4-CHOK1 cells by the pHrodo method and is superior to the control molecule.

[0141] Test Example 5. Cell Activity Experiment of ADC Molecules The purpose of this experiment was to detect the killing effect of Nectin4-ADC samples on cells and IC 50And to evaluate the in vitro activity of Nectin4-ADC by the maximum killing value.

[0142] T47D (human ductal breast cancer cells, ATCC® HTB-133 TM ), MDA-MB-468 (human breast cancer cells, ATCC® HTB-132 TM ), and MDA-MB-231 (human breast cancer cells, ATCC® HTB-26 TM ) cells were digested with pancreatin, neutralized with fresh medium, centrifuged at 1000 rpm, resuspended in culture medium, counted, and then the density of the cell suspension was adjusted to 3703 cells / mL. 135 μL / well was added to 3903 96-well cell culture plates, and no cells were seeded in the 11th column but only 135 μL of medium was added. They were cultured at 5% carbon dioxide and 37 °C for 16 hours.

[0143] ADC-1 and ADC-2 were serially diluted 5-fold with PBS using the concentration of the sample stock solution as the initial concentration to a total of 8 concentrations. The cell culture plates were taken out, and 15 μL of 10× concentration solution was added to each well. They were cultured at 5% carbon dioxide and 37 °C for 6 days.

[0144] 70 μL of CTG was added to each well, incubated at room temperature in the dark for 10 minutes, a white bottom film was attached to the bottom of the cell culture plate, and the chemiluminescence value was read on Victor3. The data of this experiment were processed using the data processing software GraphPad prism 5.0. The results are shown in Table 5.

[0145]

Table 7

[0146] NEC49-9-A (ADC-1) showed obvious killing effects in medium-expression cells and high-expression cells, and the killing was significantly reduced in low-expression cells and non-expression cells. This molecule has been shown to have higher selectivity and administration safety.

[0147] Test Example 6: Evaluation of In Vivo Efficacy of ADC Molecule High-Expression CDX Model Estrogen tablets (0.36 mg / tablet) were subcutaneously inoculated into the left posterior back of each Balb / c nude mouse. Three days later, 0.2 mL (10×10 6 cells) of T47D cells (with Matrigel, volume ratio 1:1) were subcutaneously inoculated into the right posterior back of each mouse. When the average tumor volume reached approximately 150 mm 3 , starting from this point, the mice were divided into a total of 5 groups with 8 mice / group and administration began.

[0148] The ADC compound (prepared with PBS) was intravenously injected at a dose of 10 μL / g body weight, once per mouse, for a total of 3 times. The blank solvent group was injected with PBS.

[0149] The tumor volume and body weight were measured twice a week, and the data were recorded. The data were recorded using Excel statistical software such that the average value was calculated as avg, the SD value was calculated as STDEV, and the SEM value was calculated as STDEV / SQRT (the number of animals in each group). The data were plotted using GraphPad Prism software and statistically analyzed using Two-way ANOVA or One-way ANOVA.

[0150] Calculation formula for tumor volume (V): V = 1 / 2 × L 長 × L 短 2 The relative tumor growth rate T / C (%) = (T - T0) / (C - C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment, and T0 and C0 are the tumor volumes at the start of the experiment.

[0151] Tumor inhibition rate TGI (%) = 1 - T / C (%).

[0152] The results are shown in Table 6.

[0153]

Table 8

[0154] In this experiment, the target ADC-3 and ADC-1 showed significant inhibition of the growth of human breast cancer T47D cell mouse transplanted tumors at each dose, and the dose-dependence was obvious. At the same dose, ADC-1 had a better inhibitory effect on tumor growth than the control molecule.

[0155] Test Example 7. Toxicity Experiment of ADC Molecules in Rats The ADC compound (prepared with physiological saline) was injected into SD rats (male, Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) at a dose of 5 mL / kg of body weight, once a week for a total of 3 times, via the tail vein, with 4 rats in each group. Experimental design:

[0156]

Table 9

[0157] As a result, in Group A, animal deaths occurred after the first administration, food intake stopped, and body weight decreased significantly. Therefore, the experiment was stopped and the animals were dissected, revealing adrenal hypertrophy and intestinal weight accumulation. In Group B, no clinical abnormalities were observed in the animals after 3 consecutive administrations, and no abnormalities were found after dissection. No abnormalities were observed in the blood coagulation function in each group. The results are shown in Table 8.

[0158]

Table 10

[0159] Conclusion: In the ADC-3 group, animal deaths occurred after the first administration, and there were abnormalities in multiple hematological and blood biochemical indexes. In the ADC-1 group, no abnormalities were observed clinically or after dissection in the animals after 3 consecutive administrations, no abnormalities were observed in the blood coagulation function, and except for obvious thymus shrinkage, no obvious abnormalities were found in other indexes.

[0160] The results of the primary rat toxicity experiment suggest that ADC-1 has better in vivo safety, lower toxicity and side effects compared to the control molecule.

[0161] Test Example 8. Pharmacokinetic Experiment of ADC Molecule F344 RG rats (male, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were intravenously injected once at a dose of 5 mL / kg and 10 mg / kg body weight, 4 rats in each group. 0.2 mL of whole blood was collected at 5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days before and after administration. Without adding anticoagulants, the blood samples were left standing at 4°C for 30 minutes after collection, centrifuged at 1000 g for 15 minutes, and the supernatant (serum) was taken and placed in an EP tube and stored at -80°C. The antibody (total antibody) concentration and the complete ADC concentration in the serum were measured by ELISA. The results are shown in Table 9. Results of the experiment:

Table 11

[0162] In the rat body, ADC-1 has relatively good plasma stability.

[0163] III. Formulations The equipment used in the preparation and detection of the formulations and the calculation method of the results are as follows.

[0164] 1) SEC Size Exclusion Chromatography: An analytical method for separating solutes based on the correlation between the pore size of the gel pores and the coil size of the polymer sample molecules. SEC% (content percentage of SEC monomer) = 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 the peak areas of all peaks.) △SEC% indicates the difference between the detected item after the sample is left standing under each condition and the start of standing. Equipment for SEC measurement: Agilent 1260, Column: waters, XBrige BEH200Å SEC (300×7.8mm 3.5μm)

[0165] 2) R-CE Capillary Gel Electrophoresis: It is electrophoresis performed by moving a gel as a support medium in a capillary, and is a method of separating by the molecular weight of a sample at a constant voltage. Purity percentage of 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 the peak areas of all peaks.) Equipment for R-CE measurement: Beckman model number plus800 △R-CE% indicates the difference between the detection item after leaving the sample under each condition and the start of leaving.

[0166] 3) Measurement of turbidity: The degree to which light is obstructed when passing through the aqueous layer indicates the ability of the aqueous layer to scatter and absorb light, which is not only related to the content of suspended substances, but also related to the components, sizes, shapes of the particles and the reflection characteristics of their surfaces. By comparing the absorption values of the same concentration and the same wavelength (near-ultraviolet and visible light wavelength regions) of the same protein sample, it was shown that the higher the absorption value, the higher the turbidity and the more obvious the aggregation tendency of the protein molecules in the sample. The measuring instrument was a multifunctional plate reader (Molecular Devices M5), and the absorbance values were read by putting the same volume of the sample into a 96-well plate.

[0167] 4) Measurement of osmotic pressure: The osmotic pressure was measured by the freezing point method. Based on the fact that the freezing point depression value is directly proportional to the molar concentration of the solution, a high-sensitivity temperature measuring element was used to measure the freezing point of the solution and convert it to osmotic pressure in terms of electrical quantity. The equipment manufacturer is Luser Loser, and the model number is OM815.

[0168] 5) Measurement of protein concentration: The concentration of the antibody-drug conjugate in the present disclosure was measured by the concentration of the protein, that is, the concentration of the antibody part in the antibody-drug conjugate.

[0169] The toxin in the antibody-drug conjugate absorbs at the protein characteristic absorption wavelength of 280 nm, and at the same time the toxin also absorbs at 370 nm. Therefore, the concentration of the above protein was calculated by the following formula. [Number] In the formula, A 280 nm: The average value of the absorbance of a single sample of the test solution at a wavelength of 280 nm when the optical path length is 1 cm, A 370 nm: The average value of the absorbance of a single sample of the test solution at a wavelength of 370 nm when the optical path length is 1 cm, E mAb-280 : The mass absorption coefficient of the protein at a wavelength of 280 nm, 1.532 g -1 cm -1 L, and E drug-280 : The mass absorption coefficient of the toxin at a wavelength of 280 nm, 5.17 g -1 cm -1 L, and E drug-370 : The mass absorption coefficient of the toxin at a wavelength of 370 nm, 17.89 g -1 cm -1 L, and R: The ratio of the toxin absorption coefficients at 370 nm and 280 nm, which is 3.46, C mAb : The concentration of the protein, in mg / mL, l: The optical path length, in cm (here, the optical path is 1 cm). When the test solution is diluted, the protein concentration is [Number] where N is the dilution factor. Instrument for measuring protein concentration: UV-visible spectrophotometer, model number: Nano Drop 2000.

[0170] Formulation Example 1. Screening of pH and buffer system A formulation containing the buffer system shown in Table 10, ADC-4 with a protein concentration of 20 mg / mL, and polysorbate 80 (PS80) at 0.4 mg / mL was prepared. A forced degradation study was conducted on the sample (stored at 40 °C for 1 month), and using SEC and R-CE as evaluation indicators, the effect of different buffer systems on the stability of the protein was investigated.

[0171] The results are shown in Table 10. From the SEC and R-CE data, it is shown that after storage at 40 °C for 1 month, the formulation containing His-HCl (pH 6.0) has a higher monomer purity than the other groups.

[0172]

Table 12

[0173] Formulation Example 2. Screening of Surfactants A formulation containing 10 mM His-HCl buffer at pH 6.0, ADC-4 with a protein concentration of 20 mg / mL, 80 mg / mL of sucrose, and the surfactants shown in Table 11 was prepared. A forced degradation study was conducted on the sample (shaking at 25 °C, 300 rpm for 10 days and 5 freeze-thaw cycles + standing at 25 °C for 2 days), and using appearance, SEC and R-CE as evaluation indicators, the effect of different surfactants on the stability of the protein was investigated.

[0174] The results are shown in Table 11. After forced degradation, the formulation containing 0.2 mg / mL of PS80 is clear and transparent, with a better appearance than the other groups. From the SEC and R-CE data, it is shown that there is no significant difference in the purity of the formulations with different surfactants.

[0175]

Table 13

[0176] Formulation Example 3. Screening of Buffer System Ionic Strength A formulation containing 10 mM - 30 mM of pH 6.0 His - HCl buffer, ADC - 4 with a protein concentration of 20 mg / mL, PS80 with a concentration of 0.2 mg / mL, and sucrose with a concentration of 80 mg / mL was prepared, specifically as shown in Table 12 below. A forced degradation study was conducted on the sample (shaking at 300 rpm for 10 days), and using the pH drift after replacement, appearance, SEC, and R - CE as evaluation indicators, the effect of surfactants at different concentrations on the stability of the protein was investigated.

[0177] The results are shown in Table 12. After shaking for 10 days, the formulations with ionic strengths of 10 mM and 30 mM are both clear and transparent in appearance.

[0178] [Table 14]

[0179] Formulation Example 4. Screening of Buffer System Ionic Strength A formulation containing 10 mM - 50 mM of pH 6.0 His - HCl buffer, ADC - 4 with a protein concentration of 20 mg / mL, PS80 with a concentration of 0.2 mg / mL, and sucrose with a concentration of 80 mg / mL was prepared, specifically as shown in Table 13 below. A forced degradation study was conducted on the sample (storing at 40 °C for 1 month), and using the pH drift after replacement, appearance, SEC, and R - CE as evaluation indicators, the effect of surfactants at different concentrations on the stability of the protein was investigated.

[0180] The results are shown in Table 13. The formulations with different ionic strengths are all clear and transparent in appearance. According to the SEC and R - CE data, it is shown that there is no significant difference in purity among the formulations with different buffer system ionic strengths.

[0181] [Table 15]

[0182] Formulation Example 5. Screening of Excipients and Osmotic Pressure Regulators A formulation containing 30 mM His-HCl buffer at pH 6.0, ADC-4 with a protein concentration of 20 mg / mL, and 0.2 mg / mL of PS80 was prepared. The excipients (e.g., sugars) and osmotic pressure regulators in the formulation are as shown in Table 14 below.

[0183]

Table 16

[0184] Lyoprotectant: The appearance of the lyophilized product was examined. The lyophilization procedure is as shown in Table 15 below.

[0185]

Table 17

[0186] The results are shown in Table 16. As shown by the appearance of the lyophilized solid powder, in the high sucrose concentration group, the bottom was severely concave, but the sucrose concentration was reduced to 40 mg / mL. By lyophilizing after adding glycine, the obtained solid powder was uniform and swollen without depression. It has been shown that the primary drying time required after reducing the sucrose concentration and adding glycine is shorter than the time required for the formulation using a high sucrose concentration. Preferably, it is a formulation containing 9 mg / mL of glycine.

[0187] Similarly, in the formulation with 9 mg / mL of glycine added, there was no significant difference in the appearance of the solid powder after lyophilization between the sucrose group and the α,α-trehalose dihydrate group. When sucrose is selected as the excipient and the concentration is 40 mg / mL, it is isotonic.

[0188]

Table 18

[0189] Formulation Example 6. Confirmation of the formulation A stock solution containing ADC-4 with a protein concentration of 20 mg / mL, 30 mM of pH 6.0 His-HCl, 40 mg / mL of sucrose, 0.2 mg / mL of PS80, and 9 mg / mL of glycine was prepared and lyophilized. A forced degradation stability study was conducted on the sample, and SEC and R-CE were used as evaluation indicators to examine the forced degradation stability.

[0190] The results are shown in Table 17. After standing at 40 °C for one month, there were no significant changes in either SEC or R-CE, indicating that the protein has good stability.

[0191]

Table 19

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate and a buffer, wherein the antibody-drug conjugate has the structure represented by formula NEC49-9-A: 【Chemistry 1】 It has, NEC49 is an anti-Nectin-4 antibody and comprises heavy chains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 8, 9, and 10, and light chains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 11, 12, and 13, preferably comprising a heavy chain variable region shown in SEQ ID NOs: 4 and a light chain variable region shown in SEQ ID NOs: 5, and more preferably comprising a heavy chain shown in SEQ ID NOs: 6 and a light chain shown in SEQ ID NOs:

7. n is a decimal number or integer between 1 and 10, preferably between 1 and 8, more preferably between 3 and 5. The buffer is a histidine-histidine hydrochloride buffer. The pharmaceutical composition has a pH of about 5.0 to about 6.5, preferably about 5.5 to about 6.5, and more preferably about 5.9 to about 6.

2. Pharmaceutical composition.

2. The concentration of the buffering agent is approximately 10 mM to approximately 50 mM, preferably approximately 20 mM to approximately 40 mM, and more preferably approximately 30 mM. The pharmaceutical composition according to claim 1.

3. The pharmaceutical composition further comprises a surfactant, which is preferably polysorbate, more preferably polysorbate 80 or polysorbate 20, and most preferably polysorbate 80. The pharmaceutical composition according to claim 1.

4. The concentration of the surfactant is approximately 0.05 mg / mL to approximately 0.4 mg / mL, preferably approximately 0.1 mg / mL to approximately 0.3 mg / mL, and more preferably approximately 0.2 mg / mL. The pharmaceutical composition according to claim 3.

5. The pharmaceutical composition further comprises a sugar, which is preferably selected from sucrose and α,α-trehalose dihydrate, and more preferably sucrose. The pharmaceutical composition according to claim 1.

6. The concentration of the aforementioned sugar is approximately 25 mg / mL to approximately 80 mg / mL, preferably approximately 30 mg / mL to approximately 50 mg / mL, and more preferably approximately 40 mg / mL. The pharmaceutical composition according to claim 5.

7. The pharmaceutical composition further comprises an amino acid, the amino acid being preferably glycine. The pharmaceutical composition according to claim 1.

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

9. The concentration of the antibody-drug conjugate is approximately 1 mg / mL to approximately 100 mg / mL in terms of protein concentration. Preferably, the concentration of the antibody-drug conjugate is approximately 10 mg / mL to approximately 30 mg / mL in terms of protein concentration. More preferably, the concentration of the antibody-drug conjugate is approximately 18 mg / mL to approximately 22 mg / mL in terms of protein concentration. The pharmaceutical composition according to claim 1.

10. (a) The antibody-drug conjugate having a protein concentration of approximately 10 mg / mL to approximately 30 mg / mL, (b) Polysorbate in a concentration of approximately 0.1 mg / mL to approximately 0.3 mg / mL (c) Sucrose or α,α-trehalose dihydrate in an amount of approximately 30 mg / mL to approximately 50 mg / mL, (d) Glycine in an amount of approximately 7 mg / mL to approximately 11 mg / mL, (e) Contains approximately 10 mM to approximately 50 mM histidine hydrochloride buffer, The pH of the aforementioned pharmaceutical composition is approximately 5.5 to 6.

5. Preferably, the pharmaceutical composition is (a) The antibody-drug conjugate having a protein concentration of approximately 10 mg / mL to approximately 30 mg / mL, (b) Polysorbate 80 in a concentration of approximately 0.1 mg / mL to approximately 0.3 mg / mL (c) Sucrose in a concentration of approximately 30 mg / mL to approximately 50 mg / mL (d) Glycine in an amount of approximately 7 mg / mL to approximately 11 mg / mL, (e) Contains approximately 10 mM to approximately 50 mM histidine hydrochloride buffer, The pH of the aforementioned pharmaceutical composition is approximately 5.5 to 6.

5. More preferably, the pharmaceutical composition (a) The antibody-drug conjugate having a protein concentration of approximately 10 mg / mL to approximately 30 mg / mL, (b) Polysorbate 80 in a concentration of approximately 0.1 mg / mL to approximately 0.3 mg / mL (c) Sucrose in a concentration of approximately 30 mg / mL to approximately 50 mg / mL (d) Glycine in an amount of approximately 7 mg / mL to 11 mg / mL, (e) Contains approximately 20 mM to approximately 40 mM histidine hydrochloride buffer, The pH of the aforementioned pharmaceutical composition is approximately 5.5 to 6.

5. Most preferably, the pharmaceutical composition is (a) The antibody-drug conjugate having a protein concentration of approximately 18 mg / mL to approximately 22 mg / mL, (b) Polysorbate 80 at approximately 0.2 mg / mL, (c) Sucrose at approximately 40 mg / mL, (d) Glycine at approximately 9 mg / mL, and (e) Contains approximately 30 mM histidine-histidine hydrochloride buffer, The pH of the aforementioned pharmaceutical composition is approximately 5.9 to 6.

2. The pharmaceutical composition according to claim 1.

11. A lyophilized formulation containing an antibody-drug conjugate, characterized in that it can form the pharmaceutical composition described in claim 1 after being redissolved. Freeze-dried preparation.

12. A lyophilized formulation comprising an antibody-drug conjugate, obtained by lyophilizing the pharmaceutical composition described in claim 1, Freeze-dried preparation.

13. A redissolution solution containing an antibody-drug conjugate, the redissolution solution being prepared by redissolving a lyophilized formulation containing the antibody-drug conjugate described in claim 11, Preferably, the redissolving solution consists of the following components: The pharmaceutical composition comprises (a) the antibody-drug conjugate at a protein concentration of approximately 10 mg / mL to approximately 30 mg / mL, (b) approximately 0.1 mg / mL to approximately 0.3 mg / mL of polysorbate, (c) approximately 30 mg / mL to approximately 50 mg / mL of sucrose or α,α-trehalose dihydrate, (d) approximately 7 mg / mL to approximately 11 mg / mL of glycine, and (e) approximately 10 mM to approximately 50 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is approximately 5.5 to 6.

5. More preferably, the pharmaceutical composition (a) the antibody-drug conjugate in a protein concentration of approximately 10 mg / mL to approximately 30 mg / mL, (b) polysorbate 80 in a concentration of approximately 0.1 mg / mL to approximately 0.3 mg / mL, (c) sucrose in a concentration of approximately 30 mg / mL to approximately 50 mg / mL, (d) glycine in a concentration of approximately 7 mg / mL to approximately 11 mg / mL, and (e) histidine-histidine hydrochloride buffer in a concentration of approximately 10 mM to approximately 50 mM, wherein the pH of the pharmaceutical composition is approximately 5.5 to 6.

5. Most preferably, the pharmaceutical composition is The pharmaceutical composition is characterized by comprising (a) the antibody-drug conjugate at a protein concentration of approximately 18 mg / mL to approximately 22 mg / mL, (b) approximately 0.2 mg / mL of polysorbate 80, (c) approximately 40 mg / mL of sucrose, (d) approximately 9 mg / mL of glycine, and (e) approximately 30 mM of histidine-histidine hydrochloride buffer, with a pH of approximately 5.9 to 6.

2. Reconstitution solution.

14. A kit comprising a container containing a pharmaceutical composition according to any one of claims 1 to 10, a lyophilized formulation comprising an antibody-drug conjugate according to claim 11 or 12, or a redissolving solution comprising an antibody-drug conjugate according to claim 13. kit.

15. A drug for treating a Nectin-4-mediated disease or condition, comprising a pharmaceutical composition according to any one of claims 1 to 10, a lyophilized preparation containing the antibody-drug conjugate according to claim 11 or 12, or a re-dissolving solution containing the antibody-drug conjugate according to claim 13.

16. An agent for treating viral infection, tumor, or cancer, comprising a pharmaceutical composition according to any one of claims 1 to 10, a lyophilized preparation comprising an antibody-drug conjugate according to claim 11 or 12, or a re-dissolving solution comprising an antibody-drug conjugate according to claim 13, wherein the tumor or cancer is preferably selected from urothelial carcinoma, ureteral carcinoma, breast cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, esophageal cancer, laryngeal tumor, pharyngeal tumor, oral tumor, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, and melanoma. Drugs.

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