Preparations containing anti-nectin-4 antibody-drug conjugates and uses thereof

Abstract

The present invention provides a preparation containing an anti-Nectin-4 antibody-drug conjugate (ADC) and uses thereof. The preparation comprises an ADC, a buffer, and an excipient. The anti-Nectin-4 ADC in the preparation is stable, the protein does not aggregate even after long-term storage, the small molecule drug is less likely to separate, and the osmotic pressure of the preparation is close to isotonic, facilitating targeted therapy of Nectin-4-related diseases.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This patent application claims the benefit of priority to Chinese Patent Application No. 202211341423.0, filed on October 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention belongs to the field of biological preparations, and in particular the present invention relates to preparations containing antibody-drug conjugates. [Background technology]

[0003] Antibody-drug conjugates (ADCs) are novel therapeutic agents for tumor treatment, generally consisting of an antibody or antibody-like ligand, a small molecule drug, and a linker connecting the two. ADCs combine the antitumor activity of small molecule drugs with the high selectivity of antibodies or antibody-like ligands, and are currently attracting attention in the field of tumor treatment.

[0004] Nectins (poliovirus receptor-like molecules) are a novel class of cell adhesion proteins that regulate cell-cell adhesion in cooperation with or independently of cadherins. Nectins comprise a family of four members: nectin-1, nectin-2, nectin-3, and nectin-4. All nectins have an extracellular domain containing three Ig-like loops, a transmembrane segment, and a cytoplasmic tail. Among these members, nectin-4 is specifically expressed not only in embryos and placenta but also in tumor cells, and some studies have shown that it is closely related to the development and occurrence of various tumor cells. For example, analysis of pathological sections from 2,394 tumor patients revealed that nectin-4 was widely expressed in patient populations suffering from bladder, breast, and pancreatic cancer. Therefore, nectin-4 has become an important target for the diagnosis and treatment of many tumors and cancers.

[0005] Anti-Nectin-4 antibody-drug conjugates have been prepared by chemically crosslinking anti-Nectin-4 antibodies to small molecule drugs via a linker. These ADCs utilize the antibody's targeting specificity to Nectin-4-expressing cells and its potent intracellular internalization via binding to the target antigen, combined with the effects of the small molecule drug, to achieve excellent tumor killing effects.

[0006] ADCs are typically preferentially formulated as lyophilized formulations because liquid formulations exhibit poor stability, e.g., drug release and increased aggregate formation during storage. However, both liquid and lyophilized formulations require the provision of a stabilizing formulation composition to maintain the stability of the contained ADC. Summary of the Invention

[0007] To solve the above technical problems, the present disclosure provides a formulation of a defined composition containing an anti-Nectin-4 antibody-drug conjugate.

[0008] The present disclosure provides the following technical solutions:

[0009] In one aspect, the present disclosure provides a liquid formulation comprising an anti-Nectin-4 antibody-drug conjugate or a salt thereof, a buffer, and an auxiliary material.

[0010] In the liquid formulations provided by the present disclosure, the antibody-drug conjugate or a salt thereof can be prepared according to the method described in Chinese Patent Application Publication No. 202210475286.3, and the structural proof and property evaluation are also detailed in Chinese Patent Application Publication No. 202210475286.3.

[0011] In particular, the anti-Nectin-4 antibody-drug conjugate or a salt thereof has the formula: Ab-[L-CTD] mwherein Ab represents an anti-Nectin-4 antibody or a fragment thereof, L represents a linker, CTD represents a drug, and m represents the average number of drug molecules conjugated to one Ab molecule (average DAR).

[0012] Preferably, in the antibody-drug conjugate or salt thereof, the CTD is a cytotoxic drug, and preferably, the CTD is one or more selected from the group consisting of microtubule inhibitors MMAE, DM1, DM4, tubulysin, amanitin, cachimycin, eribulin and derivatives thereof, topoisomerase inhibitors SN38, exatecan and derivatives thereof, and DNA binders PBD, doxorubicin and derivatives thereof.

[0013] m is 1.0 to 5.0, preferably 3.0 to 4.2, more preferably 3.5 to 4.5, even more preferably 3.8 to 4.2, still more preferably 3.9 to 4.1, and particularly preferably 4.0.

[0014] In the liquid formulations provided by the present disclosure, the antibody-drug conjugate or a salt thereof is represented by the following Formula I: [ka] (In the formula, Ab is an anti-Nectin-4 antibody or a fragment thereof; Ar' is any one selected from the group consisting of substituted or unsubstituted C6-C10 arylene and substituted or unsubstituted 5- to 12-membered heteroarylene, and the substitution refers to the replacement of a hydrogen atom on the group with one or more substituents selected from the group consisting of halogen (F, Cl, Br, or I), halogenated alkyl (e.g., halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, such as trifluoromethyl), and alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy); L1 is -O(CH2CH2O) linked to Ar' n-, and n is an integer ranging from 1 to 24, preferably from 1 to 10, and more preferably from 3 to 5; L2 has a structure represented by an enzyme-cleavable fragment, for example, a dipeptide, tripeptide, or tetrapeptide, or a combination of a dipeptide, tripeptide, or tetrapeptide and a self-immolative linker (i.e., a polypeptide fragment consisting of 2 to 4 amino acids, or a combination of a polypeptide fragment and a self-immolative linker), such as Val-Ala, Val-Ala-PAB, Val-Cit, Val-Cit-PAB, Phe-Lys-PAB, Ala-Ala-Ala, Gly-Gly-Phe-Gly (GGFG), MAC glucuronide phenol).

[0015] Preferably, the L2-CTD is VcMMAE, GGFG-Dxd or VC-seco-DUBA.

[0016] Preferably, when Ar' is a substituted or unsubstituted 5- to 12-membered heteroarylene, the heteroatom is N.

[0017] Preferably, Ar' is a substituted or unsubstituted C6 arylene or a substituted or unsubstituted 6-membered heteroarylene.

[0018] According to a specific embodiment of the present invention, the anti-Nectin-4 antibody-drug conjugate or a salt thereof has the following structure: [ka] It has.

[0019] Preferably, ADCs having the structure shown above have an average DAR of 3.8 to 4.2, more preferably 3.9 to 4.1, and especially preferably 4.0.

[0020] In the liquid formulation according to the present invention, the anti-Nectin-4 antibody-drug conjugate or a salt thereof comprises an anti-Nectin-4 antibody or a fragment thereof, i.e., "Ab" in the structure shown above. Antibody fragments include various functional fragments of an antibody, for example, its antigen-binding portion, such as a Fab fragment, a F(ab')2 fragment, or an scFv fragment.

[0021] In particular, the anti-Nectin-4 antibody or fragment thereof comprises three heavy chain complementarity determining regions, i.e., H-CDR1, H-CDR2, and H-CDR3, and three light chain complementarity determining regions, i.e., L-CDR1, L-CDR2, and L-CDR3; H-CDR1 comprises the amino acid sequence (DYGVS) shown in SEQ ID NO: 3, H-CDR2 comprises the amino acid sequence (VIWGGGKIYYNSVLKS) shown in SEQ ID NO: 4, H-CDR3 comprises the amino acid sequence (QGGLLFYAMDY) shown in SEQ ID NO: 5, L-CDR1 comprises the amino acid sequence (KSSQSLLNTYSQKNYLA) shown in SEQ ID NO: 8, L-CDR2 comprises the amino acid sequence (FASTRES) shown in SEQ ID NO: 9, and L-CDR3 comprises the amino acid sequence (QQHYNTPFT) shown in SEQ ID NO: 10.

[0022] Preferably, the anti-Nectin-4 antibody or a fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, and the anti-Nectin-4 antibody or a fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof.

[0023] In the context of the present disclosure, a "variant" of an amino acid sequence refers to an amino acid sequence having at least 75% sequence identity to the amino acid sequence (any percent identity equal to or greater than 75%, such as at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity).

[0024] In particular, the anti-Nectin-4 antibodies or fragments thereof defined in the present disclosure comprise at least a heavy chain variable region and a light chain variable region, both of which comprise the above-described CDRs and spaced framework regions (FRs), with these domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, with respect to the heavy chain variable region and light chain variable region contained in the anti-Nectin-4 antibodies or fragments thereof provided by the present disclosure, up to 25% of the difference in amino acid sequence resulting from "at least 75% sequence identity" may be present in any framework region in the heavy chain variable region or light chain variable region. Alternatively, with respect to the entire anti-Nectin-4 antibodies or fragments thereof provided by the present disclosure, up to 25% of the difference may be present in any domain or sequence other than the heavy chain variable region and light chain variable region in the antibodies or fragments thereof defined in the present disclosure. Differences may arise from deletions, additions, or substitutions of amino acids at any position, and substitutions may be conservative or non-conservative.

[0025] In the antibody-drug conjugates or salts thereof provided in the present disclosure, the anti-Nectin-4 antibody or fragment thereof may be in any form, such as a monoclonal antibody, single-chain antibody, bispecific antibody, single-domain antibody, nanobody, fully or partially humanized antibody, or chimeric antibody against Nectin-4. Alternatively, the antibody or fragment thereof may be a half antibody or an antigen-binding fragment of a half antibody against Nectin-4, such as a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized variable fragment (dsFv), a (disulfide-stabilized variable fragment)2 ((dsFv)2), an antigen-binding fragment (Fab), a Fab' fragment (Fab'), a F(ab')2 fragment (F(ab')2), or a variable fragment (Fv). Regarding antibody fragments provided by the present disclosure, preferably, the fragment is any fragment of an antibody capable of binding to Nectin-4. Furthermore, the nectin-4 is mammalian nectin-4, preferably primate nectin-4, more preferably human nectin-4.

[0026] Preferably, in the antibody-drug conjugate or a salt thereof defined in the present disclosure, the anti-Nectin-4 antibody or a fragment thereof may further comprise a constant region. Preferably, the anti-Nectin-4 antibody or a fragment thereof further comprises a human or mouse heavy chain constant region (CH) and / or light chain constant region (CL), more preferably a heavy chain constant region and / or a kappa or lambda light chain constant region selected from the group consisting of heavy chain constant regions of IgG, IgA, IgM, IgD, and IgE.

[0027] Preferably, the antibody is a monoclonal antibody, preferably a murine monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody, and more preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype and the light chain constant region of the monoclonal antibody is of the kappa type. Alternatively, for example, the antibody is an immunoglobulin, in particular IgA, IgD, IgE, IgG, or IgM, such as a human subtype of IgA, IgD, IgE, IgG, or IgM, more preferably a human IgG1, IgG2, IgG3, or IgG4 subtype.

[0028] According to a specific embodiment of the present invention, the anti-Nectin-4 antibody or a fragment thereof contained in the antibody-drug conjugate or a salt thereof comprises a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof.

[0029] In the context of the present disclosure, a "salt" of an anti-Nectin-4 antibody-drug conjugate refers to a pharmaceutically acceptable salt thereof, for example, an inorganic salt or an organic salt, for example, a sodium salt.

[0030] The formulation according to the present invention may be a liquid formulation, for example, in the form of a solution, emulsion, or suspension, for example, in water and / or a buffer solution. Preferably, the liquid formulation is a liquid formulation for parenteral administration. Parenteral administration may be intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, or intrasternal administration. Preferably, the liquid formulation is a liquid composition for intravenous administration, for example, intravenous infusion.

[0031] In the liquid formulation according to the present invention, the buffer is one or more selected from the group consisting of phosphate buffer, acetate buffer, and Tris-HCl buffer. Preferably, the phosphate buffer comprises a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate. Preferably, the acetate buffer comprises a mixture of acetic acid and sodium acetate.

[0032] Liquid formulations according to the present invention further comprise auxiliary materials which can function as excipients, osmolality adjusters, protein stabilizers, solubilizers, and the like.

[0033] Preferably, the auxiliary material comprises a surfactant. Preferably, the surfactant is polysorbate 20 or polysorbate 80.

[0034] Preferably, the supplemental material comprises a sugar, preferably trehalose. Optionally, the supplemental material comprises arginine.

[0035] The liquid formulation according to the present invention has a pH of 5.0 to 8.0, preferably 7.0 to 8.0, more preferably 7.0 to 7.8, for example 7.2, 7.4, 7.6, or 7.8.

[0036] Preferably, the liquid formulation according to the present invention contains the anti-Nectin-4 antibody-drug conjugate or a salt thereof at a concentration of 5 mg / mL to 40 mg / mL, preferably 8 mg / mL to 30 mg / mL, more preferably 10 mg / mL to 20 mg / mL (e.g., 10 mg / mL, 15 mg / mL, or 20 mg / mL).

[0037] Preferably, the liquid formulation according to the present invention comprises a phosphate buffer acting as a buffer system, preferably in a concentration of 5 mmol / L to 20 mmol / L, preferably 8 mmol / L to 15 mmol / L, more preferably 8 mmol / L to 10 mmol / L.

[0038] Preferably, the liquid formulation according to the present invention comprises trehalose, preferably at a concentration of 5% (w / v) to 9% (w / v), preferably 6% (w / v) to 8% (w / v), more preferably 7% (w / v).

[0039] Preferably, the liquid formulation according to the present invention contains polysorbate 20 or polysorbate 80, which acts as a surfactant, for example, at a concentration of 0.0003% (w / v) to 0.3% (w / v). Preferably, the liquid formulation contains polysorbate 20 at a concentration of 0.0003% (w / v) to 0.3% (w / v), preferably 0.01% (w / v) to 0.03% (w / v), more preferably 0.02% (w / v) to 0.03% (w / v).

[0040] In this disclosure, "w / v" refers to weight (mg) per volume (L).

[0041] More preferably, the present disclosure provides 5 mg / mL to 40 mg / mL of an anti-Nectin-4 antibody-drug conjugate or a salt thereof; 5mmol / L to 20mmol / L phosphate buffer, 5% (w / v) to 9% (w / v) of a sugar, such as trehalose, 0.0003% (w / v) to 0.3% (w / v) of a surfactant, such as polysorbate 20, and providing a formulation, e.g., a liquid formulation, comprising The liquid formulation also has a pH of 5.0 to 8.0.

[0042] More preferably, the present disclosure provides 8 mg / mL to 30 mg / mL of an anti-Nectin-4 antibody-drug conjugate or a salt thereof; 5mmol / L to 20mmol / L phosphate buffer, 6% (w / v) to 9% (w / v) of a sugar, such as trehalose, 0.01% (w / v) to 0.03% (w / v) of a surfactant, such as polysorbate 20, and providing a formulation, e.g., a liquid formulation, comprising The liquid formulation also has a pH of 7.0 to 8.0, for example, 7.2 to 7.8.

[0043] More preferably, the present disclosure provides 10 mg / mL to 30 mg / mL of an anti-Nectin-4 antibody-drug conjugate or a salt thereof; 8mmol / L to 15mmol / L phosphate buffer, 6% (w / v) to 8% (w / v) of a sugar, such as trehalose, 0.02% (w / v) to 0.03% (w / v) of a surfactant, such as polysorbate 20, and providing a formulation, e.g., a liquid formulation, comprising The liquid formulation also has a pH of 7.2 to 7.8.

[0044] More preferably, the present disclosure provides 10 mg / mL to 20 mg / mL of an anti-Nectin-4 antibody-drug conjugate or a salt thereof; 8mmol / L to 10mmol / L phosphate buffer, 6% (w / v) to 8% (w / v) of a sugar, such as trehalose, 0.02% (w / v) to 0.03% (w / v) of a surfactant, such as polysorbate 20, and providing a formulation, e.g., a liquid formulation, comprising The liquid formulation also has a pH of 7.2 to 7.6.

[0045] According to certain embodiments of the present invention, the present disclosure provides a method for manufacturing a semiconductor device comprising: 10 mg / mL of an anti-Nectin-4 antibody-drug conjugate or a salt thereof; 10 mmol / L phosphate buffer; 7% trehalose, 0.02% polysorbate 20, and providing a formulation, e.g., a liquid formulation, comprising The liquid formulation also has a pH of 7.4.

[0046] The formulation according to the present invention may be a solid formulation obtained by solidifying the liquid formulation provided by the present disclosure. Preferably, the solid formulation is a lyophilized formulation, such as a lyophilized powder injection.

[0047] According to a particular embodiment of the present invention, the solid formulation is a lyophilized formulation having a water content of 3.0% or less, preferably 1.5% or less, more preferably 1.0% or less.

[0048] Upon reconstitution of the lyophilized formulations provided by the present disclosure in a solvent (e.g., a pharmaceutically acceptable solvent such as water for injection), the resulting formulation has The anti-Nectin-4 antibody-drug conjugate or a salt thereof is contained at a concentration of 5 mg / mL to 40 mg / mL, preferably 8 mg / mL to 30 mg / mL, and more preferably 10 mg / mL to 20 mg / mL (e.g., 10 mg / mL, 15 mg / mL, or 20 mg / mL), containing a phosphate buffer at a concentration of 5 mmol / L to 20 mmol / L, preferably 8 mmol / L to 15 mmol / L, and more preferably 8 mmol / L to 10 mmol / L; Trehalose is contained in a concentration of 5% (w / v) to 9% (w / v), preferably 6% (w / v) to 8% (w / v), more preferably 7% (w / v), and / or Polysorbate 20 is contained at a concentration of 0.0003% (w / v) to 0.3% (w / v), preferably 0.01% (w / v) to 0.03% (w / v), and more preferably 0.02% (w / v) to 0.03% (w / v).

[0049] When the lyophilized formulations provided by the present disclosure are reconstituted in a pharmaceutically acceptable solvent such as water for injection, the resulting formulation has a pH of 5.0 to 8.0, preferably 7.0 to 8.0, more preferably 7.0 to 7.8, for example, 7.2, 7.4, 7.6, or 7.8.

[0050] Alternatively, a lyophilized formulation provided by the present disclosure can be reconstituted in a solvent (e.g., a pharmaceutically acceptable solvent such as water for injection) to provide a liquid formulation according to the present invention.

[0051] The lyophilized formulation provided by the present disclosure can be obtained by lyophilizing the liquid formulation according to the present invention. Preferably, the lyophilized formulation is obtained by the following steps: (1) prefreezing the liquid formulation at −40° C. or below for at least 2 hours; (2) increasing the temperature of the resulting pre-frozen product to -35°C to -20°C and annealing for at least 2 hours; (3) lowering the temperature of the obtained annealed product to −40° C. or less and pre-freezing it again for at least 2 hours; (4) increasing the temperature of the obtained pre-frozen product to -20±10°C and drying it under a pressure of 0.2 mbar or less for at least 24 hours; (5) increasing the temperature of the resulting dried product to 20±5°C and drying it under a pressure of 0.2 mbar or less for at least 10 hours; It is prepared by a method comprising:

[0052] Preferably, the method is carried out in a freeze dryer and the temperatures specified refer to the shelf temperature of the freeze dryer.

[0053] The formulation according to the present invention may be a reconstituted formulation obtained by dissolving the solid formulation provided by the present disclosure in a solvent (a pharmaceutically acceptable solvent such as water for injection). Preferably, the pharmaceutically acceptable solvent is water (e.g., water for injection) or an isotonic solution (e.g., a 0.9% NaCl solution and a 5% glucose solution).

[0054] In another aspect, the present disclosure provides use of a formulation according to the present invention (including liquid formulations, solid formulations, and reconstituted formulations) in the manufacture of a medicament for treating tumors. The medicament can be used for parenteral administration of the formulation itself, or for parenteral administration of a formulation obtained by further formulating the formulation. Parenteral administration may be intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, or intrasternal administration. Preferably, the medicament is used for intravenous administration of the formulation (e.g., intravenous infusion) or for intravenous administration of a formulation obtained by further formulating the formulation (e.g., intravenous infusion).

[0055] Alternatively, the present disclosure provides a method for treating a tumor, the method comprising administering to a subject in need of treatment (e.g., a therapeutically effective amount) of a formulation according to the present invention (including liquid formulations, solid formulations, and reconstituted formulations) or a formulation obtained by further formulating the formulation. The administration may be parenteral. Parenteral administration may be intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, or intrasternal administration. Preferably, the administration is intravenous, for example, by intravenous infusion.

[0056] The subject is a mammal, preferably a primate, more preferably a human.

[0057] Preferably, the tumor is a tumor or cancer associated with high expression of Nectin-4. More preferably, the tumor or cancer is any one selected from the group consisting of bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, prostate cancer, colorectal cancer, glioma, and mesothelioma.

[0058] The present disclosure also provides a kit comprising a formulation according to the present invention (including liquid formulations, solid formulations, and reconstituted formulations) or a formulation obtained by further formulating the formulation. The kit can be used for administering the formulation or a formulation obtained by further formulating the formulation. Optionally, the kit may also include other reagents or means for the administration described above, such as one or more of water, an isotonic solution, a syringe, etc.

[0059] Compared with the prior art, the present disclosure provides a formulation containing a novel anti-Nectin-4 antibody-drug conjugate. The anti-Nectin-4 antibody-drug conjugate in the formulation remains stable over long-term storage with minimal protein aggregation and negligible release of the small molecule drug, and the formulation has an osmotic pressure close to isotonicity. These properties collectively improve the suitability of the formulation for targeted therapy of Nectin-4-related diseases.

[0060] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0061] [Figure 1A] FIG. 1 shows the change in the main peak of HIC for formulations with different pH values ​​detected in the first screening. [Figure 1B] FIG. 1 shows the change in the main peak of SEC for formulations with different pH values ​​detected in the first screening. [Figure 2A] FIG. 1 shows the change in the main peak of HIC for formulations with different pH and different buffering agents detected in the second screening. [Figure 2B] FIG. 1 shows the change in the main peak of HIC for formulations with different pH and different ADC concentrations detected in the second screening. [Figure 2C] FIG. 1 shows the change in the main peak of SEC for formulations with different pH and different ADC concentrations detected in the second screening. [Figure 3A]FIG. 1 shows the change in the main peak of HIC for formulations with different trehalose concentrations detected in the third screening. [Figure 3B] FIG. 1 shows the changes in the main peak of HIC for formulations with different ADC concentrations and different polysorbates detected in the third screening round. [Figure 3C] FIG. 1 shows the changes in the main peak of SEC of formulations with different types and concentrations of auxiliary materials detected in the third screening. [Figure 4] FIG. 1 shows freeze-drying curves obtained in the validation of the freeze-drying process provided in the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention will now be described with reference to specific examples, which will be understood by those skilled in the art as being merely illustrative of the present invention and are not intended to limit the scope of the present invention in any way.

[0063] All experimental procedures in the following examples are conventional unless otherwise specified. All raw materials and reagents used in the following examples are commercially available unless otherwise specified.

[0064] In the present disclosure, the following detection methods are used. I.SEC-HPLC detection High-performance liquid chromatography equipment: Waters Chromatography column: Tosoh Corporation, TSKgel G3000SWXL Mobile phase: 100 mmol / L phosphate buffer (PB), 200 mmol / L L-arginine hydrochloride, and 5% isopropanol (pH 6.8) Sample preparation and analysis: The sample is diluted to 1.0 mg / mL with the mobile phase and centrifuged at 12000 rpm for 10 minutes, and the resulting supernatant is then collected for analysis. Analysis conditions: Flow rate: 0.6mL / min Column temperature: 30℃ Detection wavelength: 280 nm System suitability test: Analyze 20 μL of the working standard sample under analytical conditions. The monomer peak should show 5000 or more theoretical plates.

[0065] (II) RP-HPLC detection High-performance liquid chromatography equipment: Waters Chromatography column: Waters Acquity UPLC BEH-C18 (1.7 μm, 2.1 mm × 50 mm) Mobile phase A: 0.1% phosphoric acid aqueous solution Mobile phase B: Acetonitrile-0.1% phosphoric acid aqueous solution Blank diluent: A mixture of DMSO, purified water, and acetonitrile (volume ratio 1:1:2) Preparation of standards: Small molecule compounds MMAE, L20E and BL20E are used as standards to detect the content of free small molecule drugs. Accurately weigh 10 mg of each of the MMAE, L20E, and BL20E standards, dissolve them in acetonitrile, and dilute to 100 mL to obtain a mixed stock solution of standards for detecting free small molecule drugs. 400 μL of the stock solution was transferred to 100 μL of mobile phase A, mixed, and then 3.5 mL of blank diluent was added and mixed to obtain a mixed solution of MMAE, L20E, and BL20E standards (each at a concentration of 10 μg / mL) for detecting free small molecule drugs. The solution was further diluted with blank diluent to obtain a mixed solution of standards (each at a concentration of 1 μg / mL) for detecting free small molecule drugs.

[0066] To plot a standard curve, the solution is further diluted to different concentrations as shown below.

[0067] [Table 1] [ka] [ka]

[0068] Sample preparation and analysis: 200 μL of sample is added to 800 μL of acetonitrile, mixed thoroughly, centrifuged at 12000 rpm for 10 minutes, and the resulting supernatant is then taken for analysis. Analysis conditions: Flow rate: 0.5mL / min Column temperature: 60℃ Detection wavelength: 210 nm for MMAE detection, 241 nm for L20E and BL20E detection System suitability test: The chromatogram of the blank matrix should show no chromatographic peaks within the elution window corresponding to any one of the free small molecule drugs. Linearity of the standard curve: A standard curve is created by plotting the concentration of the standard substance against the peak area, and the regression equation and correlation coefficient are calculated. 2 ) is greater than 0.99.

[0069] (III) HIC-HPLC detection High-performance liquid chromatography equipment: Waters Chromatography column: Proteomix HIC Butyl-NP5 (4.6 mm x 35 mm) Mobile phase A: 0.025 mol / L sodium phosphate and 1.2 mol / L ammonium sulfate (pH 7.0) Mobile phase B: 0.025 mol / L sodium phosphate (pH 7.0) Mobile phase C: 100%IPA Sample preparation and analysis: The sample is diluted to 1.0 mg / mL with mobile phase B and centrifuged at 12000 rpm for 5 minutes, and the resulting supernatant is then collected for analysis. Analysis conditions: Flow rate: 0.8mL / min Column temperature: 25℃ Detection wavelength: 280 nm System suitability test: After equilibration, run the system by injecting mobile phase B as a blank control sample, which should show no interfering peaks within the integration window.

[0070] Example 1 Preparation of ADC An antibody-drug conjugate containing an anti-Nectin-4 humanized monoclonal antibody was prepared. The anti-Nectin-4 humanized monoclonal antibody is described in Chinese Patent Application Publication No. 202210475286.3 and is abbreviated as "hH2L1." The heavy and light chains of the antibody, as well as the variable regions and complementarity-determining regions (CDRs) therein, are shown below. > Amino acid sequence of heavy chain H2 (variable regions are italicized and underlined, CDRs are also in bold (according to the Kabat numbering scheme)) [ka] HC: SEQ ID NO: 1; VH: SEQ ID NO:2; H-CDR1: SEQ ID NO: 3; H-CDR2: SEQ ID NO: 4; H-CDR3: SEQ ID NO: 5 > Amino acid sequence of light chain L1 (variable regions are italicized and underlined, CDRs are also in bold (according to the Kabat numbering scheme)) [ka] LC: SEQ ID NO: 6; VL: SEQ ID NO: 7; L-CDR1: SEQ ID NO: 8; L-CDR2: SEQ ID NO: 9; L-CDR3: SEQ ID NO: 10

[0071] (1) Antibody reduction: The antibody protein was diluted to 10 ± 2 g / L using 10 mmol / L histidine buffer (pH 6.0 ± 0.1) and then reduced by adding a 10 ± 2 g / L aqueous solution of the reducing agent TCEP (tris(2-carboxyethyl)phosphine) at a molar ratio of 10:1 (TCEP:antibody). The antibody was reduced at 25 ± 5 °C for at least 120 min. The reaction mixture was then concentrated to 15 g / L and subjected to ultrafiltration exchange with at least seven volumes of buffer (50 mmol / L PB, 50 mmol / L NaCl, and 2 mmol / L EDTA·Na2, pH 7.0 ± 0.2).

[0072] (2) Coupling: To the reaction mixture obtained in step (1), 8.5% DMA (dimethylacetamide) was added as a presolvent. Next, a DMA solution containing linker-payload BL20E was added at a molar ratio of 4.8±0.3:1 (linker-payload:antibody), and the resulting reaction mixture was stirred at 25±5°C for at least 60 minutes.

[0073] (3) Ultrafiltration: The reaction mixture obtained in step (2) was subjected to ultrafiltration exchange using 7 or more volumes of a buffer solution (50 mmol / L PB, pH 7.8±0.2) to remove free small molecule drugs and residual DMA.

[0074] (4) Hydrolysis: The temperature of the reaction system obtained in step (3) was raised to 35±2° C., and then the reaction system was incubated for at least 120 minutes.

[0075] (5) Hydrophobic chromatography: Butyl Sepharose 4FF packing material or any other packing material with equivalent specifications was used. The conductivity of the hydrolysis solution obtained in step (4) was adjusted with 50 mmol / L PB and 3 mol / L ammonium sulfate, and after thorough mixing, the solution was filtered. The column packed with the packing material was equilibrated with 50 mmol / L PB and 0.45 mol / L ammonium sulfate (pH 7.5 ± 0.2), and then the solution was loaded. A linear gradient elution was performed using 0% to 80% 50 mmol / L PB (pH 7.5 ± 0.2) with a linear elution length of 10 to 12 CV. The sample peak was collected.

[0076] An anti-Nectin-4 antibody-drug conjugate having the following structure was obtained. [ka]

[0077] The average DAR of the resulting ADCs was measured and the results are shown in Table 1.

[0078] [Table 2]

[0079] Example 2 First Screening of ADC Formulations (pH) In this example, the accelerated stability of formulations with different pH values ​​was studied to identify a suitable pH range for formulations containing the ADC prepared in Example 1.

[0080] Research design: pH range: 5.0 to 8.0 Temperature: 25±2℃ Detection method: SEC-HPLC and HIC-HPLC

[0081] The pH of a protein formulation is important for product stability and biological activity. The selection of pH for a protein formulation is primarily influenced by the physicochemical properties of the protein molecule. At pH values ​​near the isoelectric point (pI), protein molecules tend to precipitate. While a solution pH near the pI can cause protein precipitation, a pH above the pI results in a strongly alkaline solution that is unsuitable for protein storage. Therefore, pH conditions below the pI require evaluation. On the other hand, excessive acidity can impair protein stability and cause the release of small molecule drugs.

[0082] Testing revealed that the ADC provided by the present disclosure has an isoelectric point of 8.0. Therefore, pH screening was performed within the range of 5.0 to 8.0. In accordance with ICH stability testing guidelines, a temperature of 25±2°C was selected for this accelerated stability study.

[0083] The formulations tested are shown in Table 2.

[0084] [Table 3]

[0085] Samples were collected and analyzed at different time points. The experimental results demonstrated that during the 10-day accelerated study, 1) the change in the area percentage of the main peak by HIC decreased with increasing pH (shown in Figure 1A and Table 3), indicating decreased release of the small molecule drug and improved formulation stability; and 2) the purity by SEC decreased with increasing pH (shown in Figure 1B and Table 3), although the change was minor and remained within an acceptable range within the pH range of 7.0–8.0.

[0086] When developing antibody-drug conjugate formulations, it is necessary to consider the pH-dependent stability of both components (high molecular weight protein and small molecule drug). Therefore, based on the results of the pH screening, it was decided to conduct the second screening at pH 7.0-8.0.

[0087] [Table 4]

[0088] Example 3 Second Screening of ADC Formulations (ADC Concentration, Buffer and pH) In this example, the accelerated stability of formulations with different ADC concentrations, buffers, and pH values ​​was studied to identify suitable concentrations, buffers, and pH ranges for formulations containing the ADC prepared in Example 1.

[0089] Research design: pH range: 7.0 to 8.0 ADC concentration: 10mg / mL~20mg / mL Buffers: Tris and PB Temperature: 25±2℃ Detection methods: SEC-HPLC, HIC-HPLC, and RP-HPLC

[0090] In Example 2, a pH range of 7.0 to 8.0 was identified. The Tris and phosphate buffer (PB) systems are compatible with this pH range. Taking into account the pH buffering capacity and buffering range of the two systems, the pH screening range was fine-tuned to 7.0 to 7.8. At the same time, to investigate the relationship between ADC concentration and product quality, the ADC concentration range was set to 10 mg / mL to 20 mg / mL, a concentration range that can meet the dosage requirements for future clinical use while preventing excessive dosing.

[0091] The formulations tested are shown in Table 4.

[0092] [Table 5]

[0093] The experimental results demonstrated the following: 1) In the PB and Tris buffer systems, the change in the area percentage of the main peak in HIC decreased with increasing pH, and the release rate of the small molecule drug also gradually decreased. A slight difference was observed between pH 7.4 and pH 7.8 (Figure 2A and Table 5). However, pH 7.8 is close to the pI (8.0) of the ADC, which poses a risk of protein precipitation. Therefore, pH 7.4 was selected for the next research stage. 2) Little difference was observed between the PB and Tris buffer systems. However, because the pH of Tris buffer is more temperature-sensitive, PB was selected as the buffer for the next research stage. 3) At ADC concentrations ranging from 10 mg / mL to 20 mg / mL, the change in the release rate of the small molecule drug detected by RP-HPLC was small, but the area percentage of the main peak in SEC tended to decrease with increasing ADC concentration (Figures 2B and 2C and Table 5).

[0094] Based on a comprehensive analysis of the above results, a successful formulation containing PB (pH 7.4) and an ADC concentration of 10 mg / mL was identified and used as the basis for the next third round of screening.

[0095] [Table 6]

[0096] Example 4 Third Screening of ADC Formulations (Supporting Materials) In this example, the accelerated stability of formulations containing different types and amounts of auxiliary materials was studied to identify auxiliary materials (including protectants and surfactants) suitable for formulations containing the ADC prepared in Example 1.

[0097] Research design: ADC concentration: 10mg / mL~20mg / mL Trehalose concentration: 5% to 9% Arginine concentration: 1% to 5% Polysorbate 20 concentration: 0.01% to 0.03% Temperature: 25±2℃ Detection methods: SEC-HPLC, HIC-HPLC, and RP-HPLC

[0098] Trehalose is an excipient and stabilizer commonly used in injectable formulations for osmotic adjustment and protein protection and stabilization during lyophilization. Polysorbate 20 acts as a stabilizer in injectable biological preparations, effectively reducing protein aggregation and improving the long-term storage stability of pharmaceuticals. Arginine functions as a protectant and effectively inhibits protein aggregation. Because the formulation must maintain a physiological osmolality range while meeting the excipient concentration requirements of lyophilized formulations, we selected a test concentration range of 5% to 9% trehalose and 1% to 5% arginine. Furthermore, because most pharmaceutical formulations contain polysorbate 20 (if present) at concentrations between 0.0003% (w / v) and 0.3% (w / v), we selected a test concentration range of 0.01% to 0.03% polysorbate 20 by referring to commercially available antibody formulations.

[0099] The formulations tested are shown in Table 6.

[0100] [Table 7]

[0101] The experimental results demonstrated the following: 1) In the accelerated process, when trehalose was added in an amount of 5% to 9%, the purity by SEC and the release rate of small molecule drugs showed little difference (as shown in Table 7 and Figure 3A), which resulted in the principle of minimal auxiliary material (the minimal auxiliary material principle). 2) As the arginine concentration increased, the release rate of the small molecule drug increased, and the SEC results showed that arginine had little effect on aggregate formation (as shown in Table 7 and Figure 3C). Therefore, arginine was excluded to adhere to the principle of minimal auxiliary materials. 3) As the ADC concentration increased, the release rate of the small molecule drug increased and the purity by SEC tended to decrease (as shown in Table 7 and Figures 3B and 3C). Therefore, a concentration of 10 mg / mL was selected for the ADC. 4) When 0.01% to 0.03% polysorbate 20 was included, no obvious difference was observed in the purity by SEC and the release rate of the small molecule drug (as shown in Table 7 and Figures 3B and 3C). Consequently, a moderate concentration of 0.02% polysorbate 20 was selected.

[0102] Based on a comprehensive analysis of the above results, we finally established a preferred formulation composition: 10 mg / mL ADC, 10 mmol / L PB, 7% trehalose, 0.02% polysorbate 20, pH 7.4. This formulation exhibited a detected osmolality of 200 mOsmol / kg to 300 mOsmol / kg. Considering that this product is administered intravenously in an isotonic compatible solution, such a composition does not significantly affect the osmolality upon administration, making the established formulation composition scientifically feasible.

[0103] [Table 8]

[0104] The process validation batch stock solution was prepared according to the formulation composition (10 mg / mL ADC, 10 mmol / L PB, 7% trehalose, 0.02% polysorbate 20, pH 7.4), and the long-term stability of the stock solution was studied at storage temperature (below -60°C).

[0105] The results shown in Table 8 indicated that the stock solution with this composition maintained good stability even after 24 months of storage. Therefore, this formulation is stable and feasible.

[0106] [Table 9]

[0107] Example 5 Establishment of the freeze-drying process and preparation of freeze-dried formulations I. Development of Freeze-Drying Process Parameters a) Determination of the main temperatures Several key temperatures in the freeze-drying process (eutectic temperature, glass transition temperature, and collapse temperature) have a significant impact on product quality, so these key temperatures need to be determined before establishing the freeze-drying process.

[0108] Eutectic temperature (Te): When a certain temperature is reached, the composition of the liquid phase in the mixed solution becomes identical overall to the composition of the solid phase that is formed, and the temperature at this point is the eutectic temperature of the solution.

[0109] Glass transition temperature (Tg): During freezing, the remaining solution becomes increasingly concentrated as ice crystals form, until a certain concentration is reached at which point the remaining water no longer crystallizes, at which point the solution reaches the temperature corresponding to the maximum freeze-concentration state.

[0110] Collapse temperature (Tc): During freeze-drying, when the temperature of the dried layer rises to a certain point, the ice in the material sublimes, and the dried layer exhibits a temperature-dependent porous honeycomb and spongy structure. When the temperature of the solid matrix in the structure rises and the rigidity of the solid matrix falls below a threshold, the vapor diffusion channel closes, and the temperature at this point is called the collapse temperature.

[0111] The three key temperatures in lyophilization were measured using a freeze-drying microscope, cryogenic differential scanning calorimetry (DSC), and a eutectic probe. The three temperatures for the liquid formulation established in Example 4 were determined to be the eutectic temperature: −18.3° C., the glass transition temperature: −31.2° C., and the collapse temperature: −27.2° C. The results are shown in Table 9.

[0112] [Table 10]

[0113] b) Determining the pre-freezing conditions The prefreezing process requires consideration of the prefreezing rate, prefreezing temperature, and prefreezing time. The prefreezing rate affects crystal size, sublimation rate, and protein activity. For example, a slow freezing rate results in larger crystals, a faster sublimation rate, and less protein damage. A slow freezing mode, i.e., a mode in which the sample is cooled along with the freeze-dryer shelf, was adopted for the liquid formulations provided in the present disclosure. Generally, the prefreezing temperature is 20°C lower than the eutectic temperature. The eutectic temperature of the formulation was detected as -18.3°C. Considering the performance of various freeze-dryers and the influence of radiant heat in the process, the prefreezing shelf temperature was set to -40°C or lower, and the prefreezing time was set to at least 2 hours.

[0114] Annealing is a process in which the temperature of a frozen product is raised to a temperature below the eutectic temperature at a specific heating rate, maintained at that temperature for a certain period of time, and then lowered to the pre-freezing temperature at a specific cooling rate. Introducing an annealing step into the pre-freezing process can enhance crystallization and remove crystalline components with low Tg values, thereby improving the overall Tg and shortening the primary drying time. The formulations provided herein contain trehalose, which has a low collapse temperature and requires a long primary drying time. Therefore, an annealing step was added to the pre-freezing process. Furthermore, the annealing temperature is generally above the glass transition temperature and below the eutectic temperature. The glass transition temperature of the formulation was detected as -31.2°C. Taking into account the difference between the shelf temperature and the actual temperature of the sample and the influence of radiant heat in the pre-freezing process, the shelf temperature for annealing was set to -35°C to -20°C and maintained at this temperature for at least 2 hours, and then the shelf temperature was lowered to -40°C or below and maintained at this temperature for at least 2 hours.

[0115] c) Determining the sublimation conditions The sublimation process involved two stages: primary drying and secondary drying.

[0116] The primary drying stage requires temperature and pressure control. Because the primary drying stage removes most of the free moisture in the sample, the rate of primary drying affects the freeze-drying process time. The rate of primary drying can be improved by increasing the freeze-drying temperature and pressure. During the freeze-drying process, the actual product temperature must be kept below the product's collapse temperature. Because sublimation is an endothermic process, the product temperature must be lower than the shelf temperature during the initial stage of sublimation. The sublimation rate can be improved by increasing the shelf temperature; therefore, the shelf temperature must be set higher than the product's collapse temperature, and the actual sublimation interface temperature must be lower than the product's collapse temperature. The collapse temperature of the formulation was detected as -27.2°C. Taking into account the performance of various freeze dryers and the effect of radiant heat during the primary drying stage, the shelf temperature for primary drying was set at -20±10°C. Furthermore, primary drying generally requires a pressure less than half the saturated vapor pressure of the product, corresponding to the product's maximum temperature. Therefore, the primary drying pressure was set at 0.2 mbar or less. Furthermore, pressure build-up testing of the primary drying stage determined that the primary drying stage should last at least 24 hours.

[0117] Secondary drying involves heating the product at a relatively high temperature to further reduce the moisture content of the freeze-dried product. Secondary drying must be performed below the denaturation temperature of the product. Therefore, the shelf temperature for secondary drying was set at 20±5°C, with the actual temperature of the sample being slightly lower than the shelf temperature. The secondary drying stage was determined to last at least 10 hours. The degree of vacuum during the secondary drying stage has little effect on product quality, so an adequate vacuum should be sufficient during the secondary drying stage.

[0118] II. Validation of the Freeze-Drying Process The freeze-drying process parameters were established as described above and are shown in Table 10.

[0119] [Table 11]

[0120] The liquid formulation established in Example 4 was freeze-dried using the following freeze-drying process to verify the freeze-drying process. The freeze-drying curve is shown in Figure 4.

[0121] Pre-freezing process: The temperature of the sample is lowered to -40°C or below and maintained for at least 2 hours; then the temperature of the sample is raised to a temperature above the glass transition temperature but below the eutectic temperature (-35°C to -20°C) and maintained for at least 2 hours to recrystallize the sample (this step can shorten the time required for primary drying); after annealing, the temperature of the sample is further lowered to -40°C or below and maintained for at least 2 hours to further maintain the rigid structure of the sample.

[0122] Primary drying stage: After the pre-freezing process was completed, the vacuum in the freeze-dryer chamber was controlled at a pressure of 0.2 mbar or less, and the temperature of the sample was raised to -20±10°C and maintained for at least 24 hours. The primary drying was stopped when the freeze-dryer chamber passed the pressure rise test (there is no fixed standard and it varies among different freeze-dryers).

[0123] Secondary drying stage: After the primary drying stage was completed, the temperature of the samples was raised to 20±5°C and maintained for at least 10 hours, after which the secondary drying stage was completed.

[0124] The experimental results showed that the freeze-dried products of the three freeze-dried batches all demonstrated a moisture content of less than 3%, good appearance, and rapid reconstitution in water for injection. The liquid formulation obtained after reconstitution contained no foreign matter and very few insoluble particulates. The detection results showed that the key quality attributes of the product remained unchanged and the consistency was good, demonstrating the feasibility of the freeze-drying process. The results are shown in Table 11.

[0125] [Table 12]

[0126] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and modifications to the present invention without departing from the spirit of the present invention, which should be included in the scope of the appended claims.

Claims

1. A liquid formulation comprising an anti-Nectin-4 antibody-drug conjugate or a salt thereof, and a buffer and auxiliary materials, wherein the anti-Nectin-4 antibody-drug conjugate or a salt thereof is represented by the formula: Ab-[L-CTD] ｍ (In the formula, Ab refers to an anti-Nectin-4 antibody or a fragment thereof comprising three heavy chain complementarity determining regions, i.e., H-CDR1, H-CDR2, and H-CDR3, and three light chain complementarity determining regions, i.e., L-CDR1, L-CDR2, and L-CDR3, wherein the H-CDR1 comprises the amino acid sequence (DYGVS) shown in SEQ ID NO: 3, and the H-CDR2 comprises the amino acid sequence (VIWGGGK) shown in SEQ ID NO:

4. the H-CDR3 comprises the amino acid sequence (QGGLLFYAMDY) shown in SEQ ID NO:5, the L-CDR1 comprises the amino acid sequence (KSSQSLLNTYSQKNYLA) shown in SEQ ID NO:8, the L-CDR2 comprises the amino acid sequence (FASTRES) shown in SEQ ID NO:9, and the L-CDR3 comprises the amino acid sequence (QQHYNTPFT) shown in SEQ ID NO:10, L represents a linker; CTD stands for drug; m represents the average number of drug molecules conjugated to one Ab molecule (average DAR).

2. In the anti-Nectin-4 antibody-drug conjugate or a salt thereof, the CTD is a cytotoxic drug, preferably one or more selected from the group consisting of microtubule inhibitors MMAE, DM1, DM4, tubulysin, amanitin, catimicin, eribulin and derivatives thereof, topoisomerase inhibitors SN38, exatecan and derivatives thereof, and DNA binders PBD, doxorubicin and derivatives thereof; 2. The liquid formulation according to claim 1, wherein m is 1.0 to 5.0, preferably 3.0 to 4.2, more preferably 3.5 to 4.5, even more preferably 3.8 to 4.2, even more preferably 3.9 to 4.1, and particularly preferably 4.

0.

3. The anti-Nectin-4 antibody-drug conjugate or a salt thereof has the following formula I: 【Chemical 1】 (In the formula, Ab is the anti-Nectin-4 antibody or a fragment thereof; Ar' is any one selected from the group consisting of substituted or unsubstituted C6-C10 arylene and substituted or unsubstituted 5- to 12-membered heteroarylene, and the substitution refers to the replacement of a hydrogen atom on the group with one or more substituents selected from the group consisting of halogen (F, Cl, Br or I), halogenated alkyl (e.g., halogenated C1-C6 alkyl, preferably halogenated C1-C4 alkyl, e.g., trifluoromethyl), and alkoxy (e.g., C1-C6 alkoxy, preferably C1-C4 alkoxy, e.g., methoxy); L １ is —O(CH ２ CH ２ O) ｎ -, and n is any integer ranging from 1 to 24, preferably from 1 to 10, more preferably from 3 to 5; L ２ is an enzyme-cleavable fragment selected from the group consisting of a dipeptide or tripeptide or tetrapeptide or a combination of said dipeptide or tripeptide or tetrapeptide with a self-immolative linker, such as Val-Ala, Val-Ala-PAB, Val-Cit, Val-Cit-PAB, Phe-Lys-PAB, Ala-Ala-Ala, Gly-Gly-Phe-Gly (GGFG), MAC glucuronide phenol; Preferably, L ２ -CTD is VcMMAE, GGFG-Dxd or VC-seco-DUBA, Preferably, when Ar' is a substituted or unsubstituted 5- to 12-membered heteroarylene, the heteroatom is N; The liquid formulation according to claim 1 or 2, characterized in that it has a structure represented by the formula: Preferably, Ar' is a substituted or unsubstituted C6 arylene or a substituted or unsubstituted 6-membered heteroarylene.

4. The anti-Nectin-4 antibody-drug conjugate or a salt thereof has the following structure: 【Chemistry 2】 The liquid formulation according to any one of claims 1 to 3, characterized in that it has

5. the anti-Nectin-4 antibody or a fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 2 or a variant thereof, and the anti-Nectin-4 antibody or a fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 7 or a variant thereof; The liquid formulation according to any one of claims 1 to 4, wherein the anti-Nectin-4 antibody or a fragment thereof preferably comprises a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, and the anti-Nectin-4 antibody or a fragment thereof preferably comprises a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 6 or a variant thereof.

6. the liquid formulation is in the form of a solution, emulsion or suspension; Preferably, the liquid formulation is a liquid composition for parenteral administration, The liquid formulation according to any one of claims 1 to 5, characterized in that the liquid formulation is preferably a liquid composition for intravenous administration, for example for intravenous infusion.

7. In the liquid formulation, the buffer is one or more selected from the group consisting of a phosphate buffer, an acetate buffer, and a Tris-HCl buffer, preferably the phosphate buffer comprises a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, and preferably the acetate buffer comprises a mixture of acetic acid and sodium acetate; Preferably, the liquid formulation further comprises an auxiliary material, Preferably, the auxiliary material comprises a sugar, preferably trehalose; Preferably, the auxiliary material comprises a surfactant, preferably polysorbate 20 or polysorbate 80; 7. The liquid formulation according to any one of claims 1 to 6, characterized in that the liquid formulation has a pH of 5.0 to 8.0, preferably 7.0 to 8.0, more preferably 7.0 to 7.8, such as 7.2, 7.4, 7.6 or 7.

8.

8. the liquid formulation contains the anti-Nectin-4 antibody-drug conjugate or a salt thereof at a concentration of 5 mg / mL to 40 mg / mL, preferably 8 mg / mL to 30 mg / mL, more preferably 10 mg / mL to 20 mg / mL; Preferably, said liquid formulation comprises said phosphate buffer acting as a buffer system, preferably said liquid formulation comprises said phosphate buffer in a concentration of 5 mmol / L to 20 mmol / L, preferably 8 mmol / L to 15 mmol / L, more preferably 8 mmol / L to 10 mmol / L; Preferably, the liquid formulation comprises trehalose, preferably the liquid formulation comprises trehalose at a concentration of 5% (w / v) to 9% (w / v), preferably 6% (w / v) to 8% (w / v), more preferably 7% (w / v); 8. A liquid formulation according to any one of claims 1 to 7, characterized in that the liquid formulation preferably comprises polysorbate 20 or polysorbate 80, for example polysorbate 20 or polysorbate 80 in a concentration of 0.0003% (w / v) to 0.3% (w / v), preferably the liquid formulation comprises polysorbate 20 in a concentration of 0.0003% (w / v) to 0.3% (w / v), preferably 0.01% (w / v) to 0.03% (w / v), more preferably 0.02% (w / v) to 0.03% (w / v).

9. The liquid formulation 5 mg / mL to 40 mg / mL of the anti-Nectin-4 antibody-drug conjugate or a salt thereof; 5 mmol / L to 20 mmol / L of the phosphate buffer; 5% (w / v) to 9% (w / v) of the sugar, for example trehalose; 0.0003% (w / v) to 0.3% (w / v) of the surfactant, for example, polysorbate 20; Including, and the liquid formulation has a pH of 5.0 to 8.0; Or, the liquid formulation is 8 mg / mL to 30 mg / mL of the anti-Nectin-4 antibody-drug conjugate or a salt thereof; 5 mmol / L to 20 mmol / L of the phosphate buffer; 6% (w / v) to 9% (w / v) of the sugar, for example trehalose; 0.01% (w / v) to 0.03% (w / v) of the surfactant, for example, polysorbate 20; Including, and the liquid formulation has a pH of 7.0 to 8.0, for example 7.2 to 7.8; Or, the liquid formulation is 10 mg / mL to 30 mg / mL of the anti-Nectin-4 antibody-drug conjugate or a salt thereof; 8 mmol / L to 15 mmol / L of the phosphate buffer; 6% (w / v) to 8% (w / v) of the sugar, for example trehalose; 0.02% (w / v) to 0.03% (w / v) of the surfactant, for example, polysorbate 20; Including, and the liquid formulation has a pH of 7.2 to 7.8; Or, the liquid formulation is 10 mg / mL to 20 mg / mL of the anti-Nectin-4 antibody-drug conjugate or a salt thereof; 8 mmol / L to 10 mmol / L of the phosphate buffer; 6% (w / v) to 8% (w / v) of the sugar, for example trehalose; 0.02% (w / v) to 0.03% (w / v) of the surfactant, for example, polysorbate 20; Including, and the liquid formulation has a pH of 7.2 to 7.6; Or, the liquid formulation is 10 mg / mL of the anti-Nectin-4 antibody-drug conjugate or a salt thereof; 10 mmol / L of the phosphate buffer; 7% trehalose, 0.02% polysorbate 20, Including, A liquid formulation according to any one of claims 1 to 8, characterized in that the liquid formulation has a pH of 7.

4.

10. A solid formulation obtained by solidifying the liquid formulation according to any one of claims 1 to 9, Preferably, the solid formulation is a freeze-dried formulation, such as a freeze-dried powder injection.

11. 11. A solid formulation according to claim 10, characterized in that the solid formulation has a water content of not more than 3.0%, preferably not more than 1.5%, more preferably not more than 1.0%.

12. A reconstituted formulation obtained by dissolving the solid formulation according to claim 10 or 11 in a solvent.

13. Use of a formulation according to any one of claims 1 to 12 in the manufacture of a medicament for treating a tumor.

14. 13. A method for treating a tumor, comprising administering to a subject in need thereof the formulation of any one of claims 1 to 12, or a formulation obtained by further formulating the formulation of any one of claims 1 to 12.

15. A kit comprising the formulation according to any one of claims 1 to 12, or a formulation obtained by further formulating the formulation according to any one of claims 1 to 12.

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