Pharmaceutical composition

Abstract

The present invention relates to pharmaceutical compositions comprising antibody drug conjugates. Also described herein are methods of making pharmaceutical compositions, kits comprising the compositions, containers holding the compositions, and uses of the pharmaceutical compositions as medicaments and for treating diseases, such as cancer.

Description

Technical Field

[0001] This invention relates to novel antibody-drug conjugate (ADC) formulations. Specifically, this invention relates to pharmaceutical compositions of specific ADCs, methods for preparing said compositions, kits containing said compositions, containers holding said compositions, and the use of said pharmaceutical compositions as medicines and for treating diseases such as cancer. Background Technology

[0002] This application relates to specific pharmaceutical compositions comprising an antibody-drug conjugate targeting trophoblast cell surface antigen 2 (TROP2).

[0003] TROP2 is a cell surface glycoprotein that is highly expressed in a variety of tumors, including breast cancer, lung cancer, pancreatic cancer, ovarian cancer, and prostate cancer. This transmembrane protein plays a role in tumor cell proliferation and is a validated target for cancer therapy.

[0004] Many antibody-drug conjugates (ADCs) targeting TROP2 are known and have shown clinical activity in patients with TROP2-expressing tumors. DS-1062 is an ADC originally developed by Daiichi Sankyo using its DXd technology. Trodelvy ® Goxatozumab is the first FDA-approved anti-TROP2 ADC for the treatment of adult patients with unresectable locally advanced or metastatic triple-negative breast cancer (TNBC) who have received two or more prior systemic therapies (at least one of which treats metastatic disease). ® Trodelvy is also approved in the United States for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma (UC) who have previously received platinum-based chemotherapy and programmed death receptor-1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitors. ® It consists of a fully humanized hRS7 IgG1κ antibody targeting TROP2 conjugated to the active metabolite SN-38 of irinotecan.

[0005] Further TROP2-targeting therapies are needed, and the applicant has developed a novel TROP2-targeting ADC with improved properties. The ADC is published in PCT international application number PCT / CN2023 / 107444, the entire contents of which are incorporated herein by reference. The ADC utilizes a stable linker to link with a next-generation camptothecin analog and is prepared using a ligase-dependent coupling technology platform. The ADC has been tested, and preclinical data indicate that it is compatible with existing ADCs (such as DS-1062 and Trodelvy). ®Compared to other methods, ADCs offer superior linker stability, superior in vitro and in vivo therapeutic effects, and superior safety.

[0006] Despite significant technological advancements that have facilitated the discovery of novel ADCs, developing these conjugates into suitable and acceptable dosage forms for therapeutic purposes remains challenging, and the formulation development process is extremely complex.

[0007] Antibodies are not only prone to degradation reactions such as oxidation, deamidation, and fragmentation, but also to particle formation and aggregation. Furthermore, the properties of antibodies often change after drug conjugation, presenting additional challenges. Antibody-drug conjugates (ADCs) are complex engineered therapeutic agents and possess more heterogeneous structures than unconjugated antibodies. This complexity makes it difficult to develop viable drug compositions, and many formulations of given ADCs, especially water-based compositions, exhibit instability over extended periods and / or under stress conditions, attributed to the multiple possible degradation pathways present in such compositions. Degradation can involve aggregation, precipitation, adsorption of molecules at the water-air interface or at the contact surface of any packaging material, improper osmotic pressure control and regulation, oxidation, photo-oxidation, hydrolysis, improper pH stability and maintenance, protein fragmentation, and / or protein unfolding. Compared to antibodies, the chemically-based cytotoxic loadings and linkers used in ADCs present different physicochemical challenges, further increasing the complexity of formulation development, such as loading stability and shedding.

[0008] Any, some, or all of the above factors may cause the drug to fail to become a drug (due to potential safety risks) or cause the drug's performance to fluctuate and become unpredictable, especially considering that drug batches may be exposed to stressful environments during preparation, transportation, and storage.

[0009] Therefore, there is a persistent need for novel and improved pharmaceutical compositions containing ADCs for the treatment of diseases such as cancer, and the present invention was designed in view of the above considerations. Summary of the Invention

[0010] In a first aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate (ADC) and a buffer, wherein the ADC has the following structure: Where X is and A mixture; and A is the anti-TROP2 antibody GQhRS7 or a fragment thereof; The linker and the antibody or antibody fragment are linked to at least one light chain of the antibody or antibody fragment via an amide bond; The pharmaceutical composition is a liquid and has a pH of 5.4 to 6.5.

[0011] The ADC shown above is an anti-TROP2 ADC, which consists of an antibody or fragment thereof targeting TROP2 (GQhRS7), wherein at least one light chain of the antibody or fragment thereof is coupled to two cytotoxic load molecules via a linker. Suitably, the linker and the antibody or fragment thereof are linked by an amide bond at the C-terminus of at least one light chain of the antibody or fragment thereof.

[0012] In one embodiment, A is an anti-TROP2 antibody, and each light chain of the antibody is linked to a linker connected to two cytotoxic load molecules via an amide bond at the C-terminus of the light chain. It should be understood that in this embodiment, the ADC therefore consists of an antibody targeting TROP2 (GQhRS7), wherein the first light chain of said antibody is coupled to two cytotoxic load molecules via linkers, and the second light chain of said antibody is also coupled to two other cytotoxic load molecules via separate but identical linkers.

[0013] It has been found that the pharmaceutical compositions according to the first aspect of the invention provide certain crucial advantages for the development of viable ADC formulations. By controlling the pH of the composition, when formulated into a liquid, it has been found that ADC loading desorption and ADC aggregation can be minimized, and the charge heterogeneity of the composition can be maintained in a more stable state.

[0014] In a second aspect of the invention, a method for preparing a pharmaceutical composition according to the first aspect is provided.

[0015] In a third aspect of the invention, a lyophilized formulation comprising an ADC and a buffer is provided, wherein the formulation, upon reconstitution, is capable of forming a pharmaceutical composition according to the first aspect.

[0016] In a fourth aspect of the invention, a method for preparing a lyophilized formulation according to the third aspect is provided.

[0017] In a fifth aspect of the invention, a container is provided for containing a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect, wherein the container can be a vial, a disposable vial, a light-proof vial, an ampoule, a syringe, a pre-filled syringe, an injection pen, or an intravenous infusion bag.

[0018] In a sixth aspect of the invention, a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect is provided for use as a medicine.

[0019] In a seventh aspect of the invention, a method for treating a subject with cancer or an autoimmune disease is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect. In a suitable embodiment, the cancer is a TROP2-associated tumor. In a suitable embodiment, the selected cancer is selected from fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma. Papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

[0020] In an eighth aspect of the invention, a combination kit is provided, the combination kit comprising a pharmaceutical composition according to the first aspect or a lyophilized formulation according to the third aspect in a container, and optionally a set of instructions having guidance on the administration of the pharmaceutical composition or lyophilized formulation.

[0021] Preferred, suitable, and optional features of any particular aspect of the invention are also preferred, suitable, and optional features of any other aspect. Attached Figure Description

[0022] Figure 1 The ADC is shown, and the ADC Trodelvy is compared. ® Trop2 binding affinity curve of antibody GQhRS7.

[0023] Figure 2 The effects of various concentrations of ADC, comparing ADC DS-1062a and antibody GQhRS7 on the viability of human pancreatic cancer cells BxPC-3, are shown as a percentage of the control group.

[0024] Figure 3 The effects of various concentrations of ADC, comparing ADC DS-1062a and antibody GQhRS7 on the activity of FaDu pharyngeal squamous cell carcinoma cells, are shown as a percentage of the control group.

[0025] Figure 4The effects of various concentrations of ADC, comparing ADC DS-1062a and antibody GQhRS7 on the viability of gastric cancer cells NCI-N87, are shown as a percentage of the control group.

[0026] Figure 5 The comparison of ADC at 0.5 mg / kg, 1.5 mg / kg, and 4.5 mg / kg with 4.5 mg / kg Trodelvy is shown when administered intravenously to mice carrying MDA-MB-468 Trop-2 positive human breast cancer xenografts. ® The inhibitory effect of DS-1062a and solvent on tumor volume. Detailed Implementation

[0027] The following provides specific implementation schemes to illustrate the technical content of this document. Through the content disclosed in this specification, those skilled in the art can readily understand other advantages and effects of this document. This document can also be implemented or applied through other different specific implementation schemes. Those skilled in the art can make various modifications and changes without departing from the spirit of this document. definition

[0028] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The term "technique" as used herein refers to those techniques commonly understood in the art, including variations and equivalents that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be readily understood by one of ordinary skill in the art, the following definitions are provided for the purpose of better illustrating this document. When a trade name appears herein, it refers to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference.

[0029] As used herein, the term "antibody" includes both whole antibodies and fragments of antibodies, provided they possess the desired biological activity, such as the ability to bind TROP2. Suitably, the antibody is GQhRS7, which is an antibody containing a heavy chain variable region (V... H ) and light chain variable region (V L Anti-TROP2 antibody, of which: The V H Include: (i) HCDR1 containing the amino acid sequence NYGMN (SEQ ID NO: 4); (ii) HCDR2 containing the amino acid sequence WINTYTGEPTYTDDFKG (SEQ ID NO: 5); (iii) HCDR3 containing the amino acid sequence GGFGSSYWYFDV (SEQ ID NO:6); and The V L Include: (i) LCDR1 containing the amino acid sequence KASQDVSIAVA (SEQ ID NO: 7); (ii) LCDR2 containing the amino acid sequence SASYRYT (SEQ ID NO:8); (iii) LCDR3 containing the amino acid sequence QQHYITPLT (SEQ ID NO: 9); Wherein V L The region also contains a recognition sequence for the ligase donor substrate. Suitable, the recognition sequence for the ligase donor substrate is ligated to V... L The C-terminus of the region. Suitably, the recognition sequence of the ligase donor substrate includes the sequence LPETGG (SEQ ID NO: 10). Suitably, the recognition sequence of the ligase donor substrate is ligated to the V region via a spacer. L Region. Appropriately, the connector has a sequence GA.

[0030] In one specific embodiment, the antibody GQhRS7 comprises the following heavy chain sequence (SEQ ID NO: 1) and light chain sequence (SEQ ID NO: 2): QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWG QGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1) DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG (SEQ ID NO: 2)

[0031] The term "buffer" refers to a component that resists pH changes when an acid or base is added to it. Those skilled in the art will be able to select a suitable buffer to achieve a desired pH range. Buffers are typically composed of a weak acid and its salt, or a weak base and its salt. Non-limiting examples of buffers include acetate buffers (acetic acid and sodium acetate), citrate buffers (citric acid and sodium citrate), histidine buffers (histidine and histidine hydrochloride), succinate buffers (succinic acid and sodium succinate), phosphate buffers (potassium dihydrogen phosphate and disodium hydrogen phosphate), and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane) buffers (BTM; zwitterionic buffers).

[0032] The term "drug-antibody ratio" or "DAR" refers to the average number of drug molecules conjugated to an antibody. Appropriate methods for determining the DAR of an ADC can be determined by those skilled in the art, including UV / Vis spectroscopy, hydrophobic interaction chromatography (HIC), reversed-phase HPLC (RP-HPLC), and mass spectrometry.

[0033] As used in this article, "patient" or "subject" refers to a mammal, including domesticated pets, animals kept as livestock, and zoo animals. Appropriately, a mammal is apes, such as humans. Most appropriately, "patient" or "subject" is a human.

[0034] It should be understood that references to “treatment” or “management” include the relief of symptoms that have already appeared in a condition. Therefore, “treatment” or “management” for a state, disorder, or condition includes: (1) delaying the appearance of clinical symptoms of a state, disorder, or condition in a person who may have a disorder or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; (2) suppressing the state, disorder, or condition, i.e., preventing, reducing, or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one of its clinical or subclinical symptoms; or (3) alleviating or reducing the disease, i.e. causing the disappearance of at least one of the state, disorder, or condition or its clinical or subclinical symptoms.

[0035] "Therapeutic effective amount" refers to the amount of a compound that, when administered to a mammal to treat a disease, is sufficient to achieve such a therapeutic effect. The "therapeutic effective amount" varies depending on the compound, the disease and its severity, and the age and weight of the mammal being treated.

[0036] As used herein, when the term “about” precedes a quantitative value, this teaching also includes the specific quantitative value itself, unless otherwise specified. Furthermore, unless otherwise indicated or reasonably inferred, the term “about” refers to a change of ±10% relative to the nominal value.

[0037] In various places within this specification, values ​​are disclosed in groups or ranges. It is specifically intended that this specification include all individual sub-combinations of members of such groups and ranges, as well as any combination of the individual endpoints of such groups or ranges. For example, integers in the range 0 to 40 are specifically intended to be disclosed individually as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and integers in the range 1 to 20 are specifically intended to be disclosed individually as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0038] As used herein, when a composition is described as having, including, or comprising specific components, or when a method is described as having, including, or comprising specific method steps, it is anticipated that the compositions of this teaching are also substantially composed of or comprised of the listed components, and that the methods of this teaching are also substantially composed of or comprised of the listed method steps.

[0039] The term “optional” or “optionally” means that the event described thereafter may but not necessarily occur, and the description includes the possibility that the event or situation described may or may not occur.

[0040] Unless otherwise claimed, any and all examples or exemplary language (e.g., "such as," "including," or "for example") is used only to better illustrate the teachings and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the teachings. Pharmaceutical Composition

[0041] According to a first aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising an antibody-drug conjugate (ADC) and a buffer, wherein the ADC has the following structure: Where X is and A mixture; and A is the anti-TROP2 antibody GQhRS7 or a fragment thereof; The linker and the antibody or antibody fragment are linked to at least one light chain of the antibody or antibody fragment via an amide bond; The pharmaceutical composition is a liquid and has a pH of 5.4 to 6.5.

[0042] According to an embodiment of the first aspect of the invention, the linker and the GQhRS7 antibody are linked via an amide bond to the C-terminus of at least one (suitably, each) light chain of the antibody. The ADC is prepared by site-specific coupling of the GQhRS7 antibody to the linker-load compound catalyzed by a ligase. The (glycine)3 moiety of the linker linked to the antibody is a recognition sequence of the ligase acceptor substrate, which promotes the enzymatic coupling of the linker-load compound to the GQhRS7 antibody under the catalysis of the ligase. For linkage with the (glycine)3 moiety of the linker, the GQhRS7 antibody includes a terminal modification at the C-terminus of each light chain, the terminal modification including a spacer and a corresponding recognition sequence of the ligase donor substrate. Specifically, the sequence of GQhRS7 is based on the amino acid sequence of hRS7 (sacituzumab), and GALPETGG (SEQ ID NO: 11) has been introduced into the C-terminus of each light chain, wherein GALPETGG is the recognition sequence of the ligase donor substrate, and GA is the spacer sequence. It should be understood that when the GQhRS7 antibody is coupled to the (glycine)3 moiety in the linker-load material under the catalysis of ligase, the recognition sequence of the ligase acceptor substrate and the recognition sequence of the ligase donor substrate react with each other to form the resulting sequence. When the GQhRS7 antibody is coupled to the corresponding recognition sequence (glycine)3 of the ligase acceptor substrate, the upstream peptide bond of GG in the LPETGG sequence is cleaved by sorting enzyme A, and the resulting intermediate is linked to the free N-terminus of G3 to generate a new peptide bond. The resulting sequence is LPETG3 (SEQ ID NO: 12). Suitablely, when the GQhRS7 antibody has been coupled to the (glycine)3 moiety of the linker, the light chain amino acid sequence SEQ ID NO: 2 is modified so that the terminal GG amino acid is omitted, thereby providing the following light chain sequence (SEQ ID NO: 3): DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPET(SEQ ID NO: 3)

[0043] Therefore, suitably, the antibody portion “A” in claim 1 includes SEQ ID NO: 1 and SEQ ID NO: 3.

[0044] The mean drug-antibody ratio (DAR) of an ADC is typically in the range of 2 to 4, or preferably about 3.5. It has been found that the pharmaceutical compositions according to the invention provide formulations with good stability in maintaining the DAR of the ADC. Therefore, in one embodiment, a pharmaceutical composition according to the first aspect is provided, wherein the ADC has a mean drug-antibody ratio (DAR) in the range of 2 to 4 (such as 2.5 to 4). In one embodiment, the ADC has a mean drug-antibody ratio (DAR) in the range of 3 to 4 (such as 3.1 to 3.9, 3.2 to 3.8, 3.3 to 3.7, or 3.4 to 3.6). In one embodiment, the ADC has a mean drug-antibody ratio (DAR) of about 3.5.

[0045] In one embodiment, a pharmaceutical composition according to the first aspect is provided, wherein after storing the composition at 25°C for at least 4 weeks, the ADC has the same (±0.2) DAR as the average drug-antibody ratio (DAR) at T=0 (the time when the sample began to be stored). In one embodiment, the DAR of the ADC after storing at 25°C for at least 3 months is the same (±0.1) as the DAR at T=0. In one embodiment, a pharmaceutical composition according to the first aspect is provided, wherein after storing the composition at 40°C for at least 2 weeks, the ADC has the same (±0.2) DAR as the average drug-antibody ratio (DAR) at T=0 (the time when the sample began to be stored). In one embodiment, the DAR of the ADC after storing at 40°C for at least 4 weeks is the same (±0.1) as the DAR at T=0.

[0046] In one embodiment, a pharmaceutical composition according to the first aspect is provided, wherein after storing the composition at 25°C for at least 4 weeks, the ADC has an average drug-antibody ratio (DAR) in the range of 2 to 4, and said DAR is the same as the DAR at T=0 (the time when the sample begins to be stored) (±0.2). In one embodiment, a pharmaceutical composition according to the first aspect is provided, wherein after storing the composition at 40°C for at least 4 weeks, the ADC has an average drug-antibody ratio (DAR) in the range of 3 to 4, and said DAR is the same as the DAR at T=0 (±0.1).

[0047] In one embodiment, the pharmaceutical composition is an aqueous liquid composition. In one embodiment, the buffer is present in the form of an aqueous buffer solution. In one embodiment, the buffer is an aqueous buffer solution with a pH in the range of 5.4 to 6.5.

[0048] In one embodiment, a pharmaceutical composition according to the first aspect is provided, wherein the ADC is dissolved in the pharmaceutical composition at a concentration of about 1 mg / mL to 100 mg / mL. It should be understood that the concentration refers to the concentration of the protein of the ADC dissolved in the pharmaceutical composition. Suitably, the ADC is dissolved in the pharmaceutical composition at a concentration of about 5 mg / mL to 100 mg / mL. Suitably, the ADC is dissolved in the pharmaceutical composition at a concentration of about 5 mg / mL to 50 mg / mL, 10 mg / mL to 40 mg / mL, 10 mg / mL to 30 mg / mL; more preferably, it is dissolved in the pharmaceutical composition at a concentration of about 15 mg / mL to 25 mg / mL, or 18 mg / mL to 22 mg / mL. In one embodiment, the ADC is dissolved in the pharmaceutical composition at a concentration of about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, or about 30 mg / mL. Most preferably, the ADC is dissolved in the pharmaceutical composition at a concentration of about 20 mg / mL.

[0049] It has been found that when formulated as a liquid, controlling the pH of the pharmaceutical composition can minimize both ADC loading loss and ADC aggregation. Specifically, it has been found that increased ADC aggregation is often observed when the pH is too low (pH < about 5.4), and increased loading loss from the conjugate is often observed when the pH is too high (pH > about 6.5). In one embodiment, the pharmaceutical composition is a liquid and has a pH of 5.4 to 6.5. In one embodiment, the pH of the composition is about 5.5 to 6.4, about 5.6 to 6.4, about 5.7 to 6.4, about 5.8 to 6.4, about 5.9 to 6.4, about 6.0 to 6.4, about 5.5 to 6.3, about 5.5 to 6.2, about 5.5 to 6.1, about 5.6 to 6.3, about 5.6 to 6.2, about 5.6 to 6.1, about 5.7 to 6.3, about 5.7 to 6.2, about 5.7 to 6.1, about 5.8 to 6.3, about 5.8 to 6.2, about 5.8 to 6.1, about 5.5 to 6.5, about 5.6 to 6.5, about 5.7 to 6.5, about 5.8 to 6.5, about 5.9 to 6.5, or about 6.0 to 6.5. Suitably, the pH of the composition is about 5.5 to 6.2, or more preferably about 5.8 to 6.2. Suitably, the pH of the composition is about 5.9 to 6.3.

[0050] In one embodiment, the pH of the composition is 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, or 6.5.

[0051] Furthermore, it has been found that, in terms of charge heterogeneity of the composition, the acidic and basic components increase more rapidly at lower pH conditions. Suitably, the pH of the composition is from about 6.0 to 6.2, or about 6.1. Suitably, the pH of the composition is 6.1.

[0052] Those skilled in the art will understand that the buffer present in the pharmaceutical composition according to the first aspect can be any buffer or buffer system suitable for providing a pH of about 5.4 to 6.5. Suitable buffers may include citrate buffers, succinate buffers, histidine buffers, phosphate buffers, acetate buffers, and malate buffers. In one embodiment, the buffer is selected from citrate buffers, succinate buffers, histidine buffers, and acetate buffers.

[0053] It has been found that histidine buffering systems can provide certain advantages over other buffering systems. Citrate and succinate buffers are more likely to exhibit increased ADC aggregation during storage; some compositions containing citrate buffers produce visible particulate matter under accelerated storage conditions; and some citrate and succinate buffer compositions produce higher levels of insoluble particles. Therefore, in a suitable embodiment, the buffer is a histidine buffer. In a more suitable embodiment, the buffer is a histidine buffer comprising a mixture of L-histidine and L-histidine monohydrochloride. In one embodiment, the pharmaceutical composition according to the first aspect is substantially free of citrate or succinate buffers. In another embodiment, the pharmaceutical composition according to the first aspect is free of citrate or succinate buffers.

[0054] In one embodiment, the pharmaceutical composition according to the first aspect comprises a buffer component. In the context of this document, a buffer component refers to two or more components capable of providing the pH buffering capacity of a buffer. In one embodiment, the buffer component is a weak acid (such as citric acid, acetic acid, succinic acid, or malic acid) and its conjugate salt. In one embodiment, the buffer component is a weak base (such as histidine) and its conjugate salt (such as hydrochloride). In a suitable embodiment, the buffer component is histidine and histidine hydrochloride.

[0055] In one embodiment, the buffer component is present at a concentration of about 5 mM to 50 mM. It should be understood that the concentration of the buffer component refers to the total concentration of all buffer components, such as the total concentration of a weak base and its conjugate salt. In one embodiment, the buffer component is present at a concentration of about 10 mM to 50 mM, such as about 10 mM to 40 mM, or about 10 mM to 30 mM. Suitably, the buffer component is present at a concentration of about 12 mM to 28 mM, such as about 14 mM to 26 mM, about 16 mM to 24 mM, or about 18 mM to 22 mM. More preferably, the buffer component is present at a concentration of about 20 mM.

[0056] In one embodiment, the pharmaceutical composition according to the first aspect further comprises a surfactant. Suitable surfactants include PEG-35 castor oil (Cremophor). ® EL, Kolliphor ® EL), PEG-40 hydrogenated castor oil (Cremophor) ® RH40, Kolliphor ® RH40), D-α-tocopherol PEG-1000 succinate (Vitamin E TPGS), capryloyl hexanoyl PEG-8 glyceride (Labrasol) ® PEG-32 glyceryl lauryl ester (Gelucire) ® 44 / 14), poloxamer, and polysorbate. In a suitable embodiment, the surfactant is polysorbate. In a suitable embodiment, the surfactant is PEG-60 sorbitol monostearate (polysorbate 60; Tween ® 60), PEG-80 sorbitol monooleate (polysorbate 80; Tween) ® 80; Kolliphor ® PS 80) or PEG-20 sorbitol monolaurate (polysorbate 20; Tween ® 20). In a suitable embodiment, the surfactant is polysorbate 80 or polysorbate 20. Polysorbate 80 has been found to perform slightly better than polysorbate 20 in minimizing the formation of insoluble particles in the composition. Therefore, in a more suitable embodiment, the surfactant is polysorbate 80.

[0057] In one embodiment, the pharmaceutical composition according to the first aspect comprises about 0.01% to about 0.2% w / v of a surfactant. Suitably, the composition comprises about 0.01% to about 0.1% w / v of a surfactant, more preferably about 0.02% to about 0.05% w / v of a surfactant, and even more preferably about 0.03% w / v of a surfactant. Suitably, the composition comprises about 0.01% to about 0.1% w / v of a polysorbate surfactant (such as polysorbate 80), more preferably about 0.02% to about 0.05% w / v of a polysorbate surfactant (such as polysorbate 80), and even more preferably about 0.03% w / v of a polysorbate surfactant (such as polysorbate 80).

[0058] In one embodiment, the pharmaceutical composition according to the first aspect further comprises a stabilizer. Suitable stabilizers include carbohydrate-based stabilizers, amino acid-based stabilizers (e.g., arginine, histidine, lysine, proline, taurine, or glycine), synthetic amphiphilic polymers (e.g., polyethylene glycol or polypropylene glycol), and ionic liquids (e.g., ammonium-based ionic liquids or imidazole-based salts). In one embodiment, the stabilizer is a carbohydrate-based stabilizer. Suitably, the carbohydrate-based stabilizer is selected from the group consisting of trehalose, mannitol, sucrose, maltose, lactose, fructose, xylitol, arabinitol, erythritol, sylitol, sorbitol, raffinose, lactitol, maltitol, and inositol. In one embodiment, the stabilizer is a non-reducing sugar. Suitably, the non-reducing sugar is selected from sucrose or trehalose. Sucrose has been found to perform slightly better than trehalose in minimizing the formation of insoluble particles in the composition. Therefore, in a more suitable embodiment, the stabilizer is sucrose.

[0059] In one embodiment, the pharmaceutical composition according to the first aspect comprises about 0.5% to about 10% w / v of a stabilizer. Suitably, the composition comprises about 1% to about 10% w / v of a stabilizer, such as about 2% to about 10% w / v, about 2% to about 9% w / v, or about 3% to about 9% w / v. More suitably, the composition comprises about 3% to about 8% w / v of a stabilizer, about 4% to about 8% w / v of a stabilizer, about 5% to about 7% w / v of a stabilizer, or even more suitably, about 6% w / v of a stabilizer. More suitably, the composition comprises about 3% to about 8% w / v of a non-reducing sugar stabilizer (such as sucrose), about 4% to about 8% w / v of a non-reducing sugar stabilizer (such as sucrose), about 5% to about 7% w / v of a non-reducing sugar stabilizer (such as sucrose), or even more suitably, about 6% w / v of a non-reducing sugar stabilizer (such as sucrose).

[0060] In one embodiment, the pharmaceutical composition according to the first aspect further comprises one or more pharmaceutically acceptable excipients. In one embodiment, the one or more pharmaceutically acceptable excipients are selected from tonic modifiers, antioxidants, and diluents. Tonic modifiers may include sodium chloride, dextrose, sucrose, trehalose, mannitol, glycerol, glycerol, sorbitol, and mixtures thereof. Antioxidants may include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), methionine, sodium bisulfite, sodium metabisulfite, thioglycerol, ascorbic acid, sodium ascorbate, and mixtures thereof.

[0061] In one embodiment, the pharmaceutical composition according to the first aspect comprises an ADC and a buffer, wherein the pharmaceutical composition is a liquid and has a pH of about 5.4 to 6.5; wherein the buffer is a histidine buffer, and the composition further comprises a polysorbate surfactant and a non-reducing sugar stabilizer.

[0062] In a suitable embodiment, the pharmaceutical composition according to the first aspect comprises an ADC and a buffer, wherein the pharmaceutical composition is a liquid and has a pH of about 5.4 to 6.5; wherein the buffer is a histidine buffer, and the composition further comprises polysorbate 20 or polysorbate 80 as a surfactant, and sucrose or trehalose as a stabilizer.

[0063] In one suitable embodiment, the pharmaceutical composition according to the first aspect comprises an ADC, a histidine buffer at a pH of 5.4 to 6.5, 0.01% to 0.2% w / v of polysorbate 20 or polysorbate 80, and 0.5% to 10% w / v of sucrose or trehalose. In another suitable embodiment, the pharmaceutical composition according to the first aspect comprises an ADC at a concentration of 5 mg / mL to 100 mg / mL, a histidine buffer at a pH of 5.4 to 6.5, 0.01% to 0.2% w / v of polysorbate 20 or polysorbate 80, and 0.5% to 10% w / v of sucrose or trehalose. In a suitable embodiment, the pharmaceutical composition according to the first aspect comprises an ADC at a concentration of 5 mg / mL to 50 mg / mL, a histidine buffer at a concentration of 5 mM to 50 mM and a pH of 6.0 to 6.2, 0.02% to 0.05% w / v polysorbate 20 or polysorbate 80, and 3% to 8% w / v sucrose or trehalose. In a more suitable embodiment, the pharmaceutical composition according to the first aspect comprises an ADC at a concentration of 15 mg / mL to 25 mg / mL, a histidine buffer at a concentration of 10 mM to 30 mM and a pH of 6.0 to 6.2, 0.02% to 0.05% w / v polysorbate 80, and 3% to 8% w / v sucrose. In a more preferred embodiment, the pharmaceutical composition according to the first aspect comprises an ADC at a concentration of 18 mg / mL to 22 mg / mL, a histidine buffer at a concentration of 15 mM to 25 mM and a pH of 6.1, 0.02% to 0.04% w / v polysorbate 80, and 5% to 7% w / v sucrose. In an even more preferred embodiment, the pharmaceutical composition according to the first aspect comprises an ADC at a concentration of 20 mg / mL, a histidine buffer at a concentration of 20 mM and a pH of 6.1, 0.03% w / v polysorbate 80, and 6% w / v sucrose.

[0064] In one embodiment, the pharmaceutical composition according to the first aspect has a molar osmotic concentration of about 150 mOSm / kg to 450 mOSm / kg, such as about 200 mOSm / kg to 400 mOSm / kg, or such as about 200 mOSm / kg to 300 mOSm / kg.

[0065] In one embodiment, the ADC maintains physical and / or chemical stability within the pharmaceutical composition according to the first aspect for at least four weeks when stored at 2°C-8°C or -20°C.

[0066] In the context of this article, physical stability refers to maintaining the appearance of the liquid composition and ensuring that the ADC remains dissolved in the liquid composition during the mentioned storage period. Appearance can be compared by visually inspecting for color changes and the presence of any visible particulate matter.

[0067] Chemical stability refers to the low levels of impurities that form during the mentioned storage period. Typically, these impurities are ADC-related and can be measured using appropriate techniques such as SEC, non-reducing CE-SDS, HIC, or HPLC.

[0068] The content of aggregates can be measured by SEC-HPLC. In one embodiment, when stored at 2°C-8°C or -20°C for at least four weeks, the pharmaceutical composition contains less than 5% (such as less than 4% or less than 3%) of aggregates, as determined by SEC-HPLC.

[0069] The content of acidic and / or basic components in the composition can be determined by CEX-HPLC. In one embodiment, after storage at 2°C–8°C or -20°C for at least four weeks, the increase in the content of the acidic peak in the pharmaceutical composition is less than 5% (such as less than 4%, less than 3%, less than 2%, or less than 1%), as determined by CEX-HPLC. In one embodiment, after storage at 2°C–8°C or -20°C for at least four weeks, the increase in the content of the basic peak in the pharmaceutical composition is less than 5% (such as less than 4%, less than 3%, less than 2%, or less than 1%), as determined by CEX-HPLC. In one embodiment, after storage at 25°C for at least three months, the increase in the content of the acidic peak in the pharmaceutical composition is less than 10% (such as less than 9%, less than 8%, less than 7%, or less than 6%), as determined by CEX-HPLC. In one embodiment, after storage at 25°C for at least three months, the increase in the content of the basic peak in the pharmaceutical composition is less than 12% (such as less than 11%, less than 10%, less than 9%, or less than 8%), as determined by CEX-HPLC.

[0070] In one embodiment, the purity of the pharmaceutical composition is greater than 96%, as determined by non-reducing CE-SDS, when stored at 2°C–8°C or -20°C for at least four weeks. In another embodiment, the purity of the pharmaceutical composition is greater than 95% (e.g., greater than 96%), as determined by non-reducing CE-SDS, when stored at 25°C for at least three months.

[0071] In one embodiment, when stored at 2°C–8°C or -20°C for at least four weeks, the content of free drug in the pharmaceutical composition is less than 0.5 mg / ml (such as less than 0.4 mg / ml or less than 0.3 mg / ml), as determined by RP-HPLC. In one embodiment, when stored at 25°C for at least four weeks, the content of free drug in the pharmaceutical composition is less than 0.75 mg / ml (such as less than 0.6 mg / ml or less than 0.5 mg / ml), as determined by RP-HPLC.

[0072] In one embodiment, the pharmaceutical composition is in the form of an injectable aqueous solution. Suitably, the injectable solution is sterile, isotonic, free of pyrogen contamination, and free of visible particulate matter. In one embodiment, the injectable aqueous solution is intended for parenteral administration via intravenous infusion.

[0073] In one embodiment, the pharmaceutical composition according to the first aspect is in the form of an injectable aqueous solution for administration by intravenous infusion, and said solution is substantially free of visible particulate matter. Suitably, said solution is free of visible particulate matter. Method for preparing pharmaceutical compositions

[0074] According to a second aspect of the invention, a method for preparing a pharmaceutical composition according to the first aspect is provided. The method comprises the steps of: mixing an ADC and a buffer, and optionally any one or more other components, optionally in any amount, concentration, or form; and optionally adjusting any one or more parameters related to the pharmaceutical composition, such as pH.

[0075] In one embodiment, the method includes the steps of: (i) mixing the ADC and buffer components together; (ii) adding a liquid (e.g., water) to obtain the desired concentration; (iii) adjusting the pH if necessary to ensure it is in the range of 5.4 to 6.5; and (iv) optionally adding one or more other pharmaceutically acceptable excipients selected from: surfactants, stabilizers, tension modifiers, antioxidants, and diluents.

[0076] In one embodiment, the method includes the steps of: (i) dissolving the buffer component in a liquid (e.g., water) to obtain a desired concentration; (ii) adjusting the pH of the buffer solution, if necessary, to ensure it is in the range of 5.4 to 6.5; (iii) adding the ADC to the buffer solution; and (iv) optionally adding one or more other pharmaceutically acceptable excipients selected from: surfactants, stabilizers, tension modifiers, antioxidants, and diluents.

[0077] In a suitable embodiment, the method includes dialysis. Therefore, in one embodiment, the method includes the steps of: (i) dissolving a buffer component in a liquid (e.g., water) to obtain a desired concentration, and optionally adding a stabilizer to the buffer solution; (ii) adjusting the pH of the buffer solution, if necessary, to ensure it is in the range of 5.4 to 6.5; (iii) adding a dialysis bag containing the ADC to the buffer solution; (iv) performing dialysis by changing the buffer solution once or multiple times; and (v) optionally adding one or more other pharmaceutically acceptable excipients selected from: surfactants, tension modifiers, antioxidants, and diluents to the resulting composition.

[0078] In one embodiment, the method includes the following steps: (i) dissolving a buffer component in a liquid (e.g., water) to obtain a desired concentration, and adding a stabilizer selected from sucrose and trehalose to the buffer solution; (ii) adjusting the pH of the buffer solution, if necessary, to ensure it is in the range of 5.4 to 6.5; (iii) adding a dialysis bag containing an ADC to the buffer solution; (iv) performing dialysis by changing the buffer solution once or multiple times; and (v) adding a surfactant selected from polysorbate 20 and polysorbate 80 to the resulting composition, and optionally adding one or more other pharmaceutically acceptable excipients selected from: tension modifiers, antioxidants, and diluents.

[0079] Other suitable methods may also be used to prepare pharmaceutical compositions, such as those that will be apparent to those skilled in the art, such as ultrafiltration, percolation, or tangential flow filtration.

[0080] In one embodiment, a product is provided that is obtained by or from a method of preparing the pharmaceutical composition of the second aspect. Lyophilized formulations

[0081] According to a third aspect of the invention, a lyophilized formulation comprising an ADC and a buffer is provided, wherein the formulation, upon reconstitution, is capable of forming a pharmaceutical composition according to the first aspect. Advantageously, the composition can be stored in a dried lyophilized form and then immediately reconstituted into a liquid (e.g., for intravenous infusion) before use. In one embodiment, the lyophilized formulation further comprises a stabilizer and / or a surfactant, and optionally one or more pharmaceutically acceptable excipients, such as a tonic modifier, antioxidant, or filler. The filler may include mannitol, sorbitol, glucose, glycine, hydroxyethyl starch, polyvinylpyrrolidone (PVP), or mixtures thereof. Suitably, the lyophilized formulation is a sterile powder.

[0082] In a fourth aspect of the invention, a method for preparing a lyophilized formulation according to a third aspect is provided. The method includes the step of lyophilizing or freeze-drying a pharmaceutical composition according to a first aspect. container

[0083] According to a fifth aspect of the invention, a container is provided for containing a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect, wherein the container can be a vial, a disposable vial, a light-proof vial, an ampoule, a syringe, a pre-filled syringe, an injection pen, or an intravenous infusion bag.

[0084] In one embodiment, the container is a transparent, amber, or brown container. Suitably, the container is a vial, ampoule, or syringe made of glass; suitably, the glass is borosilicate glass. Suitably, the container is made of plastic; suitably, the plastic is polyethylene, polypropylene, polyolefin, polyethylene terephthalate, vinyl acetate, or polyvinyl chloride.

[0085] In one embodiment, a container is provided for containing a pharmaceutical composition according to the first aspect, wherein the container is a vial, a disposable vial, a light-protected vial, an ampoule, a syringe, a pre-filled syringe, an injection pen, or an intravenous infusion bag. In one embodiment, a container is provided for containing a lyophilized formulation according to the third aspect, wherein the container is a vial, a disposable vial, a light-protected vial, or an ampoule. Therapeutic uses

[0086] According to a sixth aspect of the invention, a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect is provided for use as a medicine.

[0087] In one embodiment, a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect is provided for treating cancer or autoimmune diseases.

[0088] In one embodiment, a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect is provided for treating cancer. In one embodiment, the cancer is a TROP2-related tumor. In one embodiment, the TROP2-related tumor includes tumors that overexpress TROP2 (e.g., tumors marked as ++ or +++ using immunohistochemistry) or tumors containing one or more TROP2 gene mutations. In one embodiment, the selected cancer is selected from fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, breast cancer, etc. Head carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma. Suitably, the cancers are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

[0089] In one embodiment, use is provided in the preparation of a medicament for treating cancer or an autoimmune disease using a pharmaceutical composition according to the first aspect or a lyophilized preparation according to the third aspect. In one embodiment, the cancer is a TROP2-related tumor. In one embodiment, the TROP2-related tumor includes tumors that overexpress TROP2 or tumors containing one or more TROP2 gene mutations. In one embodiment, the selected cancer is selected from fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, breast cancer, etc. Head carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma. Suitably, the cancers are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

[0090] In one embodiment, a method is provided for treating a subject with cancer or an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to a first aspect or a lyophilized formulation according to a third aspect. In another embodiment, a method is provided for treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to a first aspect or a lyophilized formulation according to a third aspect. In yet another embodiment, a method is provided for treating a subject with cancer or an autoimmune disease, the method comprising the steps of: (i) reconstituted the lyophilized formulation according to a third aspect; and (ii) administering to the subject a therapeutically effective amount of the reconstituted formulation from step (i). In one embodiment, the cancer is a TROP2-associated tumor. In one embodiment, the TROP2-associated tumor includes tumors that overexpress TROP2 or tumors containing one or more TROP2 gene mutations. In one implementation, the selected cancers are chosen from fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, and breast cancer. Carcinoma of the head, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma. Suitably, the cancers are selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma. Suitably, the subject is a mammal; more suitably, the subject is a human. Application route

[0091] Treatment methods may include administering the pharmaceutical composition disclosed herein to the subject via any suitable route of administration.

[0092] The pharmaceutical compositions according to the invention can be administered via parenteral administration, for example by injection or infusion, including subcutaneous, intradermal, intramuscular, venous, arterial, intracardiac, intrasheathal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intra-articular, subarachnoid, and intrasternal routes; or by implantation of a reservoir or storage device, such as subcutaneous or intramuscular. Suitably, the pharmaceutical compositions according to the invention are administered via intravenous infusion. dose

[0093] Treatment methods typically involve administering a therapeutically effective amount of the pharmaceutical composition according to the invention to the subject.

[0094] The appropriate dosage of the pharmaceutical composition according to the invention may vary from patient to patient. Determining the optimal dosage will generally involve striking a balance between the level of therapeutic benefit and any risks or adverse side effects. The selected dosage level will depend on a variety of factors, including, but not limited to, route of administration, time of administration, rate of excretion of the compound, duration of treatment, other active agents, compounds and / or materials used in combination, severity of the condition, and the patient's race, sex, age, weight, condition, overall health status, and medical history. Although the dosage is generally chosen to achieve a concentration at the site of action that produces the desired effect without causing serious harmful or adverse side effects, the final determination of the dosage and route of administration will be made by the clinician.

[0095] Throughout the treatment process, administration can be achieved through single-dose, continuous, or intermittent administration (e.g., divided doses at appropriate time intervals). Single or multiple administrations are possible, with the dosage level and administration pattern chosen by the treating clinician. Combination reagent kit

[0096] According to an eighth aspect of the invention, a combination kit is provided, the combination kit comprising a pharmaceutical composition according to the first aspect or a lyophilized formulation according to the third aspect in a container, and optionally a set of instructions having instructions on the administration (e.g., by intravenous infusion) of the pharmaceutical composition or lyophilized formulation.

[0097] In one embodiment, a combination kit is provided, comprising a lyophilized formulation according to a third aspect in a container, a diluent for reconstituted with the lyophilized formulation, and optionally a set of instructions providing guidance on the administration of the reconstituted formulation (e.g., by intravenous infusion). In a suitable embodiment, the diluent is an aqueous sodium chloride solution. In a suitable embodiment, the diluent is sterile water. In a suitable embodiment, the diluent is an antibacterial water. Example General methods Protein concentration (UV method)

[0098] Protein concentration was measured using a UV-Vis spectrophotometer. A Nanodrop UV spectrophotometer was used, with blank calibration performed using ultrapure water. After mixing the test samples, 2.5 μL of the sample was pipetted and its absorbance at 280 nm was measured to determine the protein concentration of the sample. Purity (SEC-HPLC)

[0099] Sample purity was determined using size exclusion high-performance liquid chromatography (SEC-HPLC). The chromatographic column was a TSK gel G3000SWXL (7.8 × 300 mm); the mobile phase was 0.1 mol / L potassium dihydrogen phosphate-0.125 mol / L potassium chloride solution at pH 7.2. The test sample containing approximately 5 mg of the sample was diluted to 1 mL with ultrapure water. Detailed chromatographic conditions and parameters are shown in the table below. The percentages of monomers, aggregates, and fragments were calculated using the area normalization method. Charge variant (CEX)

[0100] Sample purity was determined using cation exchange high-performance liquid chromatography (CEX-HPLC). A YMC BioProIEX SF (4.6 × 100 mm) cation exchange column was used. The mobile phase composition was A: 40 Mm (N-morpholine) ethanesulfonic acid (MES) (pH 6.0), and B: 40 Mm MES + 100 mM NaCl (pH 6.0). The test sample was diluted with ultrapure water to approximately 2 mg / mL. The chromatographic parameters were set as follows: column temperature 45℃, sample chamber temperature 8℃, flow rate 0.8 mL / min, injection volume 50 μL, detection wavelength 280 nm, and elution time 40 min. The elution gradient is shown in the table below. The relative percentages of acidic components, the main peak, and basic components were calculated using the area normalization method. Purity (R CE-SDS)

[0101] The purity of R-CE-SDS was determined using capillary electrophoresis (Beckman Coulte PA800 Plus). The test sample was diluted to 10 mg / mL with ultrapure water. 85 μL of sample buffer (citrate-phosphate buffer) at pH 6.2 and 5 μL of β-mercaptoethanol were added to 10 μL of the diluted sample solution, and the sample was vortexed. After mixing and heating at 70°C for 5 minutes, the sample was cooled to room temperature and centrifuged at 13,000 rpm for 10 minutes before loading. The peak area percentages for LC, LC-Drug, and HC were calculated using the corrected peak areas. The sum of these three percentages represents the reduction purity. Instrument parameters are shown in the table below. Purity (NR CE-SDS)

[0102] The purity of NR-CE-SDS was determined using capillary electrophoresis (Beckman Coulte PA800 Plus). The test sample was diluted to 10 mg / ml with ultrapure water. 85 μL of sample buffer (citrate-phosphate buffer) at pH 6.2 and 5 μL of 500 mmol / L iodoacetamide (IAM) were added to 10 μL of the diluted sample solution, and the sample was vortexed. After homogenization and heating at 70°C for 5 minutes, the sample was cooled to room temperature and centrifuged at 13,000 rpm for 10 minutes before loading. The purity of the main peak was calculated as the percentage of the calibrated area of ​​the IgG main peak to the total calibrated peak area. Instrument parameters are shown in the table below. DAR value

[0103] The drug-antibody ratio (DAR) was determined using hydrophobic interaction chromatography (HIC). A Sepex Proteomix HIC Butyl NP5 column (4.6 × 35 mm, 5 μm particle size) was used. Mobile phase A consisted of 25 mM PB + 2 M ammonium sulfate at pH 7.0; mobile phase B consisted of 25 mM PB:IPA = 70:30 (v:v) at pH 7.0 ± 0.05. The test sample was diluted with pure water to approximately 5 mg / mL. The chromatographic parameters were set as follows: column temperature 35℃, sample chamber temperature 5 ± 3℃, flow rate 0.8 mL / min, injection volume 5 μL, detection wavelength 280 nm, and elution time 20 min. The peak area percentages of DAR0, DAR2, and DAR4 were calculated using area normalization, and the DAR value of the test product was finally calculated. The elution gradient was as follows: Free drug (RP-HPLC)

[0104] Free drug was detected using reversed-phase high-performance liquid chromatography (RP-HPLC). A Waters Xbridge C18 column (4.6 × 150 mm, 3.5 μm particle size) was used. Mobile phase A was 20 mM KH₂PO₄ solution (pH 5.0), and mobile phase B was acetonitrile solution. Proteins were precipitated using aqueous acetone, and the supernatant was used for free drug determination. Chromatographic conditions were set as follows: flow rate 0.8 mL / min, injection volume 10 μL, column temperature 25 °C, sample chamber temperature 4 °C, detection wavelength 264 nm, and analysis time 20 min. The content in the sample was calculated using the external standard method. The elution gradient was as follows: Binding activity (ELISA)

[0105] Binding activity was determined using enzyme-linked immunosorbent assay (ELISA). The antigen (GR1002T) was added to the ELISA plate and coated overnight at 2℃–8℃, followed by blocking with blocking solution. After washing the plates with washing buffer, reference product and test solution were added separately, and the plates were incubated at 25℃ with shaking for 1 hour. After washing the plates with washing buffer, horseradish peroxidase-labeled goat anti-human IgG-Fc antibody solution was added, and the ELISA plate was incubated at 25℃ with shaking for 1 hour. After washing the plates with washing buffer, TMB was added for color development and the plate was protected from light. After 10 minutes, the reaction was terminated with stop solution, and the absorbance was measured at a wavelength of 450 nm. OD was measured using a microplate reader. 450 And the software tools included with the ELISA reader were used to create a graph with concentration as the x-axis and OD as the y-axis. 450 The value is a four-parameter logistic curve (Y = (AD) / (1+(X / C)^B) + D) with the ordinate as the ordinate. After fitting and plotting the standard curve, the correlation coefficient R between the curve fitting of the standard and the sample is obtained. 2 and EC 50 The relative binding activity of the sample is calculated using the following formula: Relative binding activity of sample (%) = EC of standard 50 ÷Sample EC 50 ×100%. Appearance and visible foreign objects (visual inspection)

[0106] The appearance of the sample was assessed by visual inspection. The light intensity of the transparency detector was maintained between 1000 lx and 1500 lx. The sample was held at eye level and gently shaken or inverted to prevent the formation of air bubbles. Visual inspection was performed against a black and white background. Results were recorded in terms of appearance and visible foreign matter. Insoluble particles (photoresist method)

[0107] Insoluble particles were detected using an AccuSizer A2000 insoluble particle detector. The parameters were set according to the table below. osmolar concentration

[0108] An Osmomat 3000 osmotic pressure detector was used for detection. Before testing the samples, the detector was calibrated with calibration solutions (300 mOsm / L, 500 mOsm / L). Material abbreviation Example 1: Formation of antibody-drug conjugates (ADCs)

[0109] Antibody-drug conjugates (ADCs) have the following structures: 1.1 Preparation of supported materials — Option A Option A: Synthesis route of the loading material 1.1.1 Formation of N-(2-bromo-5-fluorophenyl)acetamide (compound 2)

[0110] Concentrated H₂SO₄ (3 mL) was added to a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL), followed by the addition of 2-bromo-5-fluoroaniline-compound 1 (100 g, 526.27 mmol) in portions at room temperature. The mixture was stirred for 3 hours and then poured into 2000 mL of ice water. A precipitate formed, which was collected by filtration and dried under vacuum at room temperature to give N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 1 H NMR (400 MHz, DMSO- d6 ) δ 7.68 (dd, J = 8.9, 6.0Hz, 1H), 7.61 (ddd, J = 10.7, 5.3, 3.1 Hz, 1H), 7.02 (ddd, J = 8.9, 8.0, 3.1Hz, 1H), 2.11 (s, 3H). MS m / z 232.0(M+H). 1.1.2 Formation of N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (compound 3)

[0111] At -78°C, n-BuLi (594 mL, 1.6 M n-hexane solution, 950.22 mmol) was added dropwise over 1 hour to a stirred solution of compound 2 (105 g, 452.48 mmol) in THF (1000 mL). After completion, the mixture was stirred under N2 for 0.5 hours. Then, at -78°C, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise over 0.5 hours, and the mixture was stirred at -78°C to room temperature for 6 hours. The mixture was poured into 500 mL of saturated NH4Cl aqueous solution at 0°C, extracted with ethyl acetate (500 mL × 3), washed with brine (250 mL × 2), dried over Na2SO4, and concentrated. The mixture was ground with PE / EA (1:1, 100 mL) for 10 minutes, filtered, and the filter cake was collected and vacuum dried to obtain N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g), a yellow solid. LCMS m / z 206.1 (M-18+H), 246.1 (M+Na). Option A: Synthetic route of the supported compound (compound 12) 1.1.3 Formation of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (compound 4)

[0112] Silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol) were added to a stirred mixture of compound 3 (24 g, 107.50 mmol) in CH2Cl2 (170 mL) and water (170 mL). The mixture was stirred at 30 °C for 6 hours. The mixture was filtered through diatomaceous earth and washed with CH2Cl2 (100 mL). The filtrate was concentrated and purified by FCC (EA / PE = 0%–40%) to give N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide (14 g) as a pale yellow solid. MS m / z 222.1 (M+H). 1.1.4 Formation of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (compound 5)

[0113] At 0 °C, 1-butyl nitrite (8.48 g, 63.28 mmol) was added to a stirred mixture of compound 4 (14 g, 63.28 mmol) and THF (500 mL), followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0 °C for 2 hours. After stirring, the mixture was acidified with HCl (2N) to adjust the pH to 3. The mixture was extracted with ethyl acetate (200 mL × 3), washed with brine (100 mL × 2), dried over Na₂SO₄, and concentrated under reduced pressure. The crude mixture was milled with tert-butyl methyl ether (200 mL) for 10 minutes, filtered, and the filter cake was collected and dried under vacuum to give N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (12 g) as a yellow solid. MS m / z 251.1 (M+H). 1.1.5 Formation of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (compound 6)

[0114] 10% Pd / C (1 g) was added to a solution of compound 5 (12 g, 47.96 mmol) in acetic anhydride (90 mL) and THF (90 mL), and the mixture was stirred at 25 °C under a H2 atmosphere for 16 hours. After cooling to 0 °C, Et3N (20 mL) was added dropwise, and the mixture was stirred at 0 °C for 1 hour. The mixture was then analyzed by Celite.® After filtration, the filtrate was poured into ice water (500 mL) and extracted with ethyl acetate (500 mL × 3). The extract was washed with brine (250 mL × 2), dried over Na₂SO₄, and concentrated. The residue was ground with tert-butyl methyl ether (120 mL) for 10 minutes, filtered, and the filter cake was collected and dried under vacuum to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g), a yellow solid. MS m / z 279.1 (M+H). 1.1.6 Formation of N-(8-amino-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide (compound 7)

[0115] An aqueous solution of HCl (2N, 150 mL) was added to a MeOH solution (7.9 g, 28.39 mmol), and the mixture was stirred at 50 °C for 7 hours. After cooling to 0 °C, a saturated NaHCO3 aqueous solution was added dropwise to adjust the pH to 8. The mixture was extracted with ethyl acetate (200 mL × 3), washed with brine (200 mL × 2), dried over Na2SO4, and concentrated under reduced pressure to give N-(8-amino-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide (6.0 g), a yellow solid. 1 ¹H NMR (400 MHz, chloroform-d) δ 6.57 (s, 3H), 6.18 (td, J = 11.1, 2.4 Hz, 2H), 4.52 (dt, J = 13.3, 5.0 Hz, 1H), 3.13 (ddd, J = 17.5, 13.0, 4.6 Hz, 1H), 3.00 – 2.81 (m, 1H), 2.69 (dtd, J = 9.4, 4.6, 2.5 Hz, 1H), 2.09 (s, 3H), 1.79 (qd, J = 13.0, 4.3 Hz, 1H). MS m / z 237.1 (M+H). 1.1.7 Formation of N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide (compound 8)

[0116] At 0 °C, NCS (2.26 g, 16.93 mmol) was added in portions to a DMF (80 mL) solution of compound 7 (4.0 g, 16.93 mmol), and the mixture was stirred at room temperature for 16 hours. The mixture was poured into 200 mL of ice water. A precipitate formed, which was collected by filtration and dried under vacuum at room temperature to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthyl-2-yl)acetamide (4.0 g) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 8.0 Hz, 1H), 7.71 (s, 2H), 6.62 (d, J = 11.9 Hz, 1H), 4.53 (ddd, J = 13.0, 8.0, 4.7Hz, 1H), 3.18 – 3.04 (m, 1H), 2.91 (ddd, J = 17.5, 12.4, 4.8 Hz, 1H), 2.21 –2.08 (m, 1H), 1.99 – 1.83 (m, 4H). MS m / z 271.0 (M+H). 1.1.8 Formation of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide (compound 10)

[0117] To a mixture of compound 8 (4.0 g, 14.78 mmol) in toluene (400 mL), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolazine-3,6,10(4H)-trione (4.28 g, 16.25 mmol, CAS No. 110351-94-5), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol), and o-cresol (10 mL) were added, and the mixture was heated under reflux at N2 for 24 hours. The solvent was removed under reduced pressure, and the mixture was purified by FCC (THF / CH2Cl2=0%-60%) to give N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide (4.1 g), a brown solid. MS m / z 498.1 (M+H). 1.1.9 Formation of (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione hydrochloride (compound 11)

[0118] The mixture of compound 10 (2.0 g, 4.02 mmol) in 20 mL of concentrated HCl was stirred at 70 °C and N2 for 36 hours. The mixture was concentrated under reduced pressure to give crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione hydrochloride (2 g) as a brown solid. MS (ESI) m / z 456.1 (M+H). 1.1.1 Formation of 0(9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione trifluoroacetate (compound 12).

[0119] Compound 12 was prepared from compound 11 by preparative HPLC. 1 H NMR (400 MHz, DMSO-d6) δ8.51(d, J = 4.8 Hz, 3H), 8.17 (d, J = 10.2 Hz, 1H), 7.38 (s, 1H), 6.56 (s, 1H), 5.74(d, J = 19.4 Hz, 1H), 5.52 – 5.40 (m, 3H), 5.16 (s, 1H), 3.44 (dd, J = 16.3, 4.1Hz, 1H), 3.19 (t, J = 13.9 Hz, 1H), 2.57 (d, J = 14.0 Hz, 1H), 2.26 (t, J = 14.3Hz, 1H), 1.89 (hept, J = 7.0 Hz, 2H), 0.89 (t, J= 7.3 Hz, 3H). MS m / z 456.0 (M+H). HPLC retention time: 1.395 min (Agilent 1200; column: Waters XBridge C18 4.6×50 mm, 3.5 μm; flow rate: 2.0 mL / min; gradient elution: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min-1.50 min-2.50 min-2.52 min-3.00 min; temperature: 40℃; phase: A: acetonitrile, B: H2O (0.05% TFA); wavelength: 214 nm / 254 nm). 1.2 Preparation of Linker-Loaded Intermediate 1 — Option B Option B: Synthesis route of connector-load intermediate 1

[0120] Linker-support intermediate 1 was synthesized using Rink-amide-MBHA resin via conventional solid-phase peptide synthesis. Fmoc was used to protect the amino acids in the linker unit. Coupling reagents were selected from HOBT, HOAt / DIC, DCC, EDCI, or HATU. After synthesis, the product was cleaved from the resin using a TFA / TIS / H2O solution. The product was purified by preparative HPLC, lyophilized, and stored for later use. MS m / z: [MH] - = 1382.6. 1.3 Preparation of linker-support material — Option C 1.3.1 Preparation of Compound 13

[0121] Weigh 4.33 g of Fmoc-Gly-Gly-OH and 6.84 g of Pb(OAc)4 and add them to a 500 mL single-necked round-bottom flask. Under a nitrogen atmosphere, add anhydrous THF / toluene (120 mL / 40 mL) and stir to dissolve. Then add 1.16 mL of pyridine to the reaction system. Heat the reaction system to 80 °C and reflux for 5 hours under a nitrogen atmosphere. Take a sample and detect the reaction by HPLC to monitor the reaction. Cool the reaction system to room temperature, filter, and wash the filter cake three times with EA. Combine the filtrates and concentrate to dryness. Perform column chromatography (PE:EA = 100:0-50:100) to give approximately 2000 mg of compound 13 as a white solid, in a yield of 44%. 1.3.2 Preparation of Compound 15

[0122] Compound 13 (200 mg) was weighed and added to a 100 mL single-necked round-bottom flask. 15 mL of THF was then added and stirred to dissolve. Compound 14 (312 mg, 3.0 equivalent) and TsOH·H₂O (15 mg, 0.15 equivalent) were then added to the reaction mixture. The reaction mixture was allowed to react overnight at room temperature. Samples were taken and analyzed by TLC (PE / EA = 1:1) to monitor the reaction. The reaction was quenched with saturated sodium bicarbonate solution. The mixture was extracted three times with EA. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate, and concentrated. The crude product was purified by column chromatography (PE:EA = 5:1–1:1) to give approximately 80 mg of compound 15 as a colorless oil, in a yield of 29%. MS m / z: [M+H] + = 501.1 Option C: Synthesis route of linker-support 1.3.3 Preparation of Compound 16

[0123] Compound 15 (200 mg) was weighed and added to a 100 mL single-necked round-bottom flask. Then, 10 mL of EtOH and 5 mL of EA were added and completely dissolved. Next, under a nitrogen atmosphere, 40 mg of palladium on carbon was added to the reaction system, and the system was purged three times with hydrogen. The reaction system was maintained under a hydrogen atmosphere and stirred at room temperature for 0.5 h. Samples were taken and analyzed by TLC (DCM / MeOH = 10:1) to monitor the reaction. The reaction system was filtered, and the filter cake was washed three times with EA. The filtrates were combined and concentrated to dryness to give 200 mg of product as a white solid, with a yield of 100%. The product could be used directly in the next reaction without purification. MS m / z: [MH]⁻ = 409.4. 1.3.4 Preparation of compound 21 via solid-state synthesis 1.3.4.1 Step A — Formation of Compound 17

[0124] Weigh 2.0 g of dichloropolymer resin and place it in a polypeptide synthesis tube. Add DCM (10 ml) and allow it to swell at room temperature for 30 minutes. Remove the solvent by vacuum aspiration. Wash the resin twice with DCM, 7 mL each time for 1 minute. Remove the solvent by vacuum aspiration. Then weigh compound 16 (200 mg) and add it to a 50 ml centrifuge tube. Add DCM (approximately 10 ml). Dissolve the solid by shaking. Add this to the resin. Stir to immerse all the resin in the solution (if there is resin adhering to the tube wall, wash the tube wall with a small amount of DCM). Stir for 4-5 hours. After the reaction is complete, add methanol. Stir for 30 minutes. Remove the solvent by vacuum aspiration. Wash the resin sequentially with DMF once, methanol once, DMF once, methanol once, and DMF twice, 10 mL each time for 1 minute. Remove the solvent by vacuum aspiration. Take a small amount of dried resin for ninhydrin detection. The resin is colorless and transparent, and the solution is pale yellow, which meets the requirements for the next coupling step. 1.3.4.2 Step B — Formation of Compound 18

[0125] Compound 17 was deprotected twice by adding 10 mL of 20% piperidine / DMF solution and reacting for 10 minutes each time. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each wash consisting of 10 mL of DMF for 1 minute. The solvent was removed by vacuum aspiration. A small amount of dried resin was tested for ninhydrin. Both the resin and the solution turned deep blue.

[0126] Add 563 mg Fmoc-Phe-OH and 197 mg HOBt to a 50 mL centrifuge tube. Then add approximately 7 mL of DMF. Dissolve the solid by shaking. Then add 0.24 mL of DIC. Activate for 10-30 minutes to obtain an activated reaction solution. Add 3 molar equivalents of the activated reaction solution to the resin. Stir to completely immerse the resin in the solution. Stir for 2-3 hours. After the reaction is complete, remove the solvent by vacuum suction. Wash the resin sequentially with DMF twice, methanol once, DMF once, methanol once, and DMF twice, each wash volume being 10 mL and each wash lasting 1 minute. Remove the solvent by vacuum suction. Take a small amount of dried resin for ninhydrin detection. The resin is colorless and transparent, and the solution is pale yellow, meeting the requirements for the next coupling step. 1.3.4.3 Step C — Formation of Compound 19

[0127] Compound 18 was deprotected twice by adding 10 mL of 20% piperidine / DMF solution and reacting for 10 minutes each time. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each wash consisting of 10 mL of DMF for 1 minute. The solvent was removed by vacuum aspiration. A small amount of dried resin was tested for ninhydrin. Both the resin and the solution turned deep blue.

[0128] Add 531 mg Fmoc-Gly-Gly-OH and 197 mg HOBt to a 50 mL centrifuge tube. Then add approximately 10 mL of DMF. Dissolve the solid by shaking. Then add 0.24 mL of DIC. Activate for 10-30 minutes to obtain an activated reaction solution. Add 3 molar equivalents of the activated reaction solution to the resin. Stir to completely immerse the resin in the solution. Stir for 2-3 hours. After the reaction is complete, remove the reaction solution by vacuum suction. Wash the resin twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each wash volume being 10 mL and each wash lasting 1 minute. Remove the solvent by vacuum suction. Take a small amount of dried resin for ninhydrin detection. The resin is colorless and transparent, and the solution is pale yellow, meeting the requirements for the next coupling step. 1.3.4.4 Step D — Formation of Compound 20

[0129] Compound 19 was deprotected twice by adding 10 mL of 20% piperidine / DMF solution, with each reaction lasting 10 minutes. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each wash volume being 10 mL and lasting 1 minute. The solvent was removed by vacuum aspiration. A small amount of dried resin was tested for ninhydrin. Both the resin and the solution turned deep blue. Then, 462 mg of MC-OSu was placed in a 50 mL centrifuge tube, and 10 mL of DMF was added. The solid was dissolved by shaking. Then, 0.24 mL of DIEA was added to the resin. The mixture was stirred to completely immerse the resin in the solution. The mixture was stirred for 2-3 hours. After the reaction was complete, the reaction solution was removed by vacuum aspiration. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol, and twice with DMF, each wash volume being 10 mL and lasting 1 minute. The solvent was removed by vacuum aspiration. A small amount of dried resin was tested for ninhydrin. The resin was colorless and transparent, and the solution was pale yellow, meeting the requirements for the next coupling step. 1.3.4.5 Step E — Formation of Compound 21

[0130] The resin of compound 20 was washed twice with 10 mL of methanol. The solvent was then completely removed by vacuum suction. The resin was poured out and weighed. A lysis buffer was prepared in a 250 mL Erlenmeyer flask, wherein the ratio of TFE / DCM was 80% / 20%, and the volume was 7-8 times the weight of the peptide resin. The lysis buffer was added to the peptide resin and shaken well. The resin was thoroughly immersed in the lysis buffer and lysed at room temperature for 2-3 hours. The lysis buffer was then filtered out using a simple filter made from a syringe, and the resin was washed with 1-2 mL of DCM and discarded. 150 mL of pre-cooled anhydrous diethyl ether was then added to the lysis buffer, shaken well, and allowed to stand for 20-30 minutes. The system was centrifuged at 3500 rpm for 3 minutes in a 50 mL centrifuge tube, and the supernatant was discarded. The solid was shaken with pre-cooled anhydrous diethyl ether, washed once under sonication, centrifuged at 3500 rpm for 3 minutes, and the supernatant was discarded. The solid was placed in a centrifuge tube and allowed to air dry overnight. After purification, 125 mg of compound 21 was obtained as a white solid, with a yield of 40%. MS m / z: [MH] - = 641.5. 1.3.5 Preparation of Compound 22

[0131] Weigh 150 mg of compound 21 and 55 mg of TSTU into a 10 mL single-necked round-bottom flask. Add 3 mL of anhydrous DMF under a nitrogen atmosphere and stir for 20 minutes. Then, add 18 mg of compound 12 and 20 μl of DIEA sequentially to the reaction system. Stir at room temperature and under a nitrogen atmosphere for 2–8 hours. Samples were taken and analyzed by HPLC to monitor the reaction. After the reaction was complete, the reaction system was preparatively purified, the product was collected and freeze-dried to obtain approximately 22 mg of compound 22 as a pale yellow solid. MS m / z: [M+H] + = 1081.0. 1.3.6 Preparation of Linker-Support Material

[0132] Weigh compound 22 (30 mg) and add it to a 10-inch single-necked round-bottom flask, then add purified water (2 ml). After stirring until dissolved, add 2 ml of DMF solution containing linker-supported intermediate 1 (19.5 mg) to the reaction system and stir. After reacting overnight, monitor the reaction using HPLC until all starting materials are converted to the intermediate. Add an appropriate amount of Tris-Base solution or other solution that promotes ring-opening reaction directly to the reaction mixture and continue the reaction at 0°C–40°C for 0.2–20 hours. Monitor the reaction by HPLC until all intermediate is consumed, then quench with acetic acid solution.

[0133] The reaction mixture was preparatively purified, the target product was collected and lyophilized to give approximately 25 mg of the linker-supported product as a pale yellow solid. MS m / z: [(M+3H) / 3] + = 1194.4. 1.4 Preparation of antibody GQhRS7 1.4.1 Construction of expression vector encoding antibody

[0134] The sequence of GQhRS7 is based on the amino acid sequence of hRS7, a known antibody (WO03074566A2), but GALPETGG has been introduced into the C-terminus of each light chain, where LPETGG is the recognition sequence for the ligase donor substrate and GA is the spacer sequence.

[0135] The antibody GQhRS7 contains the following heavy chain sequence (SEQ ID NO: 1) and light chain sequence (SEQ ID NO: 2): SEQ ID NO: 1: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQ GSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. SEQ ID NO: 2: DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG

[0136] To generate an expression vector encoding the light chain, the nucleic acid sequence (SEQ ID NO: 2) was cloned into the pCDNA 3.3 vector (Life technology); to generate an expression vector encoding the heavy chain, the nucleic acid sequence (SEQ ID NO: 1) was cloned into the pCDNA 3.3 vector (Life technology). 1.4.2 Antibody Expression

[0137] The light and heavy chains encoding GQhRS7 were paired and mixed at a 2:1 mass ratio. The plasmid pairs and PEIMAX (Polyscience) transfection reagent were diluted separately with HEK293F basal medium and then thoroughly mixed. Neon transfection was then performed. ® Transfection was performed via electroporation. The mixture was allowed to stand at room temperature and then added to HEK293F seeding medium. Cells were cultured at 32°C for 24 hours and samples were taken for cell density and viability analysis, with 10% volume of HEK293F feed medium added. The culture temperature was then lowered to 32°C for subsequent culture. Cell cultures were sampled again at 72 hours for cell density and viability analysis. Cell cultures were sampled again at 144 hours for cell density and viability analysis. 1.4.3 Antibody Purification

[0138] GQhRS7 was purified by affinity chromatography according to the manufacturer's instructions. In short, a column (Borglon, Shanghai, China) was packed with MabSelect SureLX resin (GE Healthcare) and equilibrated with 50 mM Tris, 150 mM NaCl, pH 7.4. The supernatant of the cell culture was then obtained and applied to the column. The column was washed with 50 mM Tris, 150 mM NaCl, pH 7.4 to remove non-specifically bound proteins. The antibody was then eluted with 50 mM citrate buffer (pH 3.5), and the antibody-containing eluent was adjusted to pH 6.5 with 1 M Tris-HCl (pH 9.0). Finally, the antibody buffer was replaced with 50 mM Tris, 150 mM NaCl, pH 7.4 using an Anicon Ultra-15 centrifuge filter (Merk Millipore). 1.5 ADC fabrication

[0139] ADCs are prepared by site-specific conjugation of GQhRS7 antibody to linker-loaded compound via ligase catalysis (for suitable conjugation methods, see WO2015165413A1).

[0140] Antibody GQhRS7 was treated by ultrafiltration, dialysis, or desalting column treatment. The storage solution was replaced with ligase buffer. The ADC was prepared by coupling GQhRS7 with the linker-load (described in Example 1.3 above) under the catalysis of wild-type sorting enzyme A or a mutant ligase optimized therefrom. The modified antibody and linker-load were thoroughly mixed in a ligase buffer at a molar ratio of 1:1 to 1:100 and then added to a solid-phase coupling system. The solid-phase coupling system included a ligase immobilized on a matrix of the solid-phase coupling system. The immobilized ligase catalyzed the coupling reaction of antibody GQhRS7 with the linker-load. The coupling reaction was carried out at 4°C–40°C for 0.5 h–20 h. After the reaction, the reaction mixture was ultrafiltered or dialyzed to remove unreacted intermediates, yielding the ADC. Store the ADC in a buffer containing 20 mM citric acid, 200 mM NaCl, and pH 5.0 at 4°C or -80°C. 1.6 Analysis and characterization of ADC HIC-HPLC Detection and Analysis of ADC

[0141] The DAR (drug-antibody ratio) distribution of the ADCs was analyzed by HIC-HPLC. The results showed that the conjugates mainly contained ADCs with a DAR of 3.4-3.5. Example 2: Biological activity of ADC 2 2.1 Human Trop 2 Combined Affinity

[0142] Human Trop 2 ECD at a concentration of 0.5 μg / mL was coated onto a 96-well plate and incubated overnight at 4°C. The plate was then blocked with 3% BSA-PBST for 1 hour at room temperature. After washing with PBST (0.05% Tween), different concentrations of ADC and Trodelvy were added. ® (Compare with Trop 2 ADC) or antibody (GQhRS7) was added to a 96-well plate and incubated at room temperature for 60 minutes. After incubation, goat anti-human FC secondary antibody (HRP) (Sinobiological, SSA001) was added at a ratio of 1:100,000 and incubated at room temperature for 60 minutes. After washing, the plate was treated with TMB solution (Sigma, T0440) as the HRP substrate, and the reaction was terminated with 1M H2SO4. The absorbance of each well was measured at 450 nm.

[0143] Figure 1 It shows ADC, Trodelvy ® Trop 2 binding affinity curves for GQhRS7. The results indicate that the ADC exhibits affinity for both GQhRS7 and Trodelvy. ® They have similar Trop 2 binding affinity. 2.2 Effects on tumor cell proliferation

[0144] Trop 2-positive cancer cells BxPC-3, FaDu, and NCI-N87 (3000 to 5000 cells) were seeded in 96-well plates and allowed to adhere overnight. Cells were treated for 168 hours with various concentrations of ADC, DS1062a (comparative Trop 2 ADC – as described in US2016 / 0297890A), or antibody (GQhRS7). Cells were analyzed using CellTiter-Globe assay. ® A luminescent cell viability assay was used to detect cell viability and calculate the percentage of cell viability.

[0145] Figures 2 to 4 Cell viability curves of ADC, DS1062a, and GQhRS7 against BxPC-3, FaDu, and NCI-N87 cells are shown. In Trop2-positive BxPC-3, FaDu, and NCI-N87 cells, the IC50 of ADC was [not specified]. 50 The values ​​are 0.226 nmM, 0.128 nmM, and 0.742 nmM, respectively, while the IC of DS1062a... 50 The concentrations were 0.692 nM, 0.170 nM, and 2.101 nM, respectively. The antibody alone (GQhRS7) did not show any significant effect on cell viability at the tested concentrations. 2.3 In the BxPC-3 / HepG2 co-culture experiment, the bystander killing effect of ADC on HepG2 was observed.

[0146] The cell concentrations of Trop2-positive BxPC-3 cells and Trop2-negative HepG2 cells were adjusted to 1×10⁻⁶. 6 Cells were seeded at 300 μL / well (BxPC-3:HepG2 = 2:1) into six-well plates, and 2.7 mL of medium containing 45% RPMI-1640 + 45% DMEM + 10% FBS was added. Cells were incubated overnight at 37°C with 5% CO2. 3 mL of 20 nM ADC and DS1062a were added to each well of the overnight cultured cells (final drug concentration 10 nM / well). A negative control group was set up: 3 mL of medium containing 45% RPMI-1640 + 45% DMEM + 10% FBS was added to each well. After treatment, cells were transferred to an incubator and incubated for 96 hours. After incubation, cells were digested, washed once with 1X PBS, and then transferred to flow cytometry tubes and centrifuged at 2000 rpm for 3 minutes. The supernatant was then discarded, and cell count and viability were assessed. A certain amount of cells was washed with 1X PBS, centrifuged, and the supernatant was discarded. 200 μL of 100 nM anti-human Trop2 antibody was added, and the cells were mixed and incubated at 4°C for 30 minutes. The cells were then washed with 1X PBS, centrifuged, and the supernatant was discarded. 200 μL of 5 μg / mL human IgG Fc antibody was added, and the resulting cells were mixed and incubated at 4°C for another 30 minutes. Finally, the cells were washed with 1X PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in PBS and analyzed by flow cytometry using FlowJo software. The results are shown in Table A, and the results indicate that the bystander killing effect of the ADC was superior to that of the comparative ADC-DS1062a. Table A: Bystander lethality test results for ADC and DS1062a 2.4 In vivo mouse xenograft tumor growth inhibition

[0147] 0.2 mL of cancer cells (human pancreatic cancer cell line BxPC-3; pharyngeal squamous cell carcinoma line FaDu; or gastric cancer cell line NCI-N87) were suspended in a suspension (cell density = 10 × 10⁻⁶). 6 Tumors were injected subcutaneously into the right scapula of 6-8 week old SPF female BALB / c nude mice, suspended in a matrix gel buffer (PBS:Matrix gel = 1:1). Tumor diameter was measured using calipers and calculated according to formula V = 0.5a × b. 2 Calculate the tumor volume (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor).

[0148] For BxPC-3 tumors, 6 days after cell seeding, when the average tumor volume was approximately 151 mm... 3 Animals were randomly divided into a solvent control group, a DS1062a 3mg / kg group, and an ADC 3mg / kg group, with 6 animals in each group.

[0149] For FaDu tumors, 11 days after cell seeding, when the average tumor volume was approximately 123 mm... 3 Animals were randomly divided into a solvent control group, a DS1062a 3mg / kg group, and an ADC 3mg / kg group, with 6 animals in each group.

[0150] For NCI-N87 tumors, 8 days after cell seeding, when the average tumor volume was approximately 188 mm... 3 Animals were randomly divided into a solvent control group, a DS1062a 3mg / kg group, and an ADC 3mg / kg group, with 6 animals in each group.

[0151] Animals in each group were administered the drug via tail vein injection, while the control group received an equal volume of solvent. Tumor volume was measured twice weekly for 35 days post-administration, and inter-group comparisons were made for tumor volume at day 35. T / C and TGI values ​​were calculated using tumor volume. The calculation formula is as follows: T / C% = T RTV / C RTV ×100% (T RTV Treatment group RTV; C RTV The relative tumor volume (RTV) was calculated based on the results of tumor measurements using the formula: RTV = V t / V0, where V0 is the average tumor volume measured at the time of grouping (i.e., day 0), V t T represents the average tumor volume at a given measurement, and T0 represents the average tumor volume at that measurement. RTV With C RTV Data from the same day were used. TGI (%) was calculated as follows: TGI (%) = [1 - (mean tumor volume at the end of treatment - mean tumor volume at the beginning of treatment) / (mean tumor volume at the end of solvent control - mean tumor volume at the beginning of solvent control)] × 100%. Results are shown in Table B1. In all three tumor types, ADC inhibited tumor growth to a greater extent than the comparative ADC – DS1062a. In the FaDu tumor cohort, all six mice achieved complete tumor regression after ADC treatment. Table B1: Inhibitory effect of ADCs on xenograft tumors in mice 2.5 ADC for MDA-MB-468 In vivo efficacy evaluation

[0152] MDA-MB-468 (Trop-2 positive human breast cancer cells; ATCC, HTB-132) cells were maintained in vitro as a monolayer in L-15 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal agent under an air atmosphere containing 0% CO2 at 37°C. Cells in the exponential growth phase were collected and counted for tumor inoculation. 0.2 mL of PBS was mixed with 10 × 10⁶ cells in a 1:1 matrix gel. 6 One MDA-MB-468 cell was subcutaneously injected into the right flank of BALB / c nude mice. After 24 days, when the average tumor volume reached 187 mm², [the cells were observed]. 3 At that time, tumor-bearing mice were divided into groups and intravenously administered 0.5 mg / kg, 1.5 mg / kg and 4.5 mg / kg of ADC, and 4.5 mg / kg of Trodelvy. ® Alternatively, administer DS1062a at 4.5 mg / kg. Measure tumor volume twice weekly using calipers. Calculate T / C and TGI values ​​using tumor volume as described in Example 2.4.

[0153] The results are shown in Table B2 and Figure 5 In the study, at equivalent doses, ADCs showed significantly better efficacy than Trodelvy. ® It is also slightly better than the DS1062a. Table B2: Inhibitory effect of ADC on xenograft tumors in MDA-MB-468 mice Example 3: Preliminary Formulation Screening 3 3.1 Formation of ADC buffer formulations 1-12

[0154] Prepare 2 L of each of the 12 buffer solutions according to Table C. Transfer the ADC sample in citrate buffer (50.1 mg / ml) to ultrafiltration centrifuge tubes (x12), then add the buffer solutions according to Table C, and replace the liquid by ultrafiltration centrifugation. Centrifuge at 3500 rpm for 15 minutes each time, ensuring a liquid replacement volume ratio of at least 8 times and a liquid replacement rate of at least 99%. After the culture medium replacement is complete, measure the protein concentration, volume, and pH, and dilute the sample to the target concentration of 20.0 mg / ml with the appropriate buffer.

[0155] On a clean workbench, aseptically fill formulation 1-12 into 2R vials (1.0 ml / vial), and then stopper and crimp the cap.

[0156] Formulations 1-12 were then stored for up to 4 weeks under various conditions (-20°C, 2°C-8°C, 25°C, and 40°C) and analyzed periodically as described in Section 3.2. Table C: Summary of Preliminary Formulations 3.2 Analysis of ADC buffer formulations 1-12

[0157] The formulation was analyzed using the techniques described in the General Methods section above. 3.3 Preliminary formulation screening results

[0158] The protein concentrations and pH values ​​of the buffer formulations after storage for 2–4 weeks under various conditions are shown in Table D. No significant changes in protein concentration or pH were observed under these conditions. Table D: Protein concentration and pH of formulations 1-12 after storage for up to 4 weeks

[0159] In terms of appearance, no obvious foreign matter was observed in any sample at T=0. Except for formulation 1, which was a colorless and transparent liquid, all other formulations were colorless and slightly milky white liquids. Formulations 4, 5, and 6 produced fine, visible particles after storage at 40°C for 4 weeks, indicating that ADCs are more prone to particle formation in citrate buffer systems. Furthermore, formulations 1 to 6 were removed, thawed at -20°C, and allowed to stand for 4 hours. Visual inspection revealed numerous small air bubbles that persisted even after four hours of standing.

[0160] The levels of insoluble particles in formulations 1-12 during stable storage under different conditions (Tables E1 and E2) were evaluated. The results showed that the number of insoluble particles in the citrate buffer (formulations 4-6) and succinate buffer (formulations 10-12) systems was relatively higher than that in the acetate buffer (formulations 1-3) and histidine buffer (formulations 7-9) systems. Table E1: Insoluble particle levels under different temperature and time conditions (A: ≥2μm; B: ≥5μm; Unit: particles / ml) Table E2: Insoluble particle levels under different temperature and time conditions (C: ≥10 μm; D ≥25 μm; Unit: particles / ml)

[0161] Based on the SEC-HPLC aggregate content results in Table F1, the aggregate amount increased in all formulations under high temperature and accelerated conditions, but the increases were larger in formulations 4-6 and 10-12, indicating that ADCs are more prone to aggregate formation in citrate and succinate buffers. After 4 weeks of storage at 2℃-8℃ and -20℃, no significant increase in aggregates was observed in any formulation. Furthermore, it was found that for a given buffer system, the higher the pH, the faster the aggregate formation rate. Table F1: Percentage of aggregates in formulations 1-12 under different temperature and time conditions (SEC-HPLC) Table F2: Monomer percentage content of formulations 1-12 under different temperature and time conditions (SEC-HPLC) Table F3: Fragment percentage content of formulations 1-12 under different temperature and time conditions (SEC-HPLC)

[0162] According to the CEX-HPLC results in Table G1, although the proportion of acidic components in formulations 1-12 increased after 4 weeks of storage at high temperature (40°C), the increase in the proportion of acidic components in formulations 7-9 was smaller compared with T=0.

[0163] According to the CEX-HPLC results in Table G2, although the proportion of the main peak in formulations 1-12 decreased after 4 weeks of storage at high temperature (40°C), the decrease in the proportion of the main peak in formulations 9 and 12 was smaller compared with T=0.

[0164] According to the CEX-HPLC results in Table G3, although the proportion of alkaline components in formulations 1-12 increased after 4 weeks of storage at high temperature (40°C), the increase in the proportion of alkaline components in formulations 9 and 12 was smaller compared with T=0.

[0165] These results indicate a significant correlation between the charge components of the ADC and the pH of the formulation. When the pH is below 6.0, both acidic and basic components increase rapidly, but when the pH is above 6.0, their increases are relatively slow. No significant changes were observed in the content of acidic components, basic components, and the main peak at 2℃–8℃ and -20℃. Table G1: Acid peak content of formulations 1-12 under different temperature and time conditions (CEX-HPLC) Table G2: Peak content of formulations 1-12 under different temperature and time conditions (CEX-HPLC) Table G3: Alkaline peak content of formulations 1-12 under different temperature and time conditions (CEX-HPLC)

[0166] According to the R-CE-SDS purity results in Table H1, the purity of each formulation did not decrease significantly under accelerated and high-temperature conditions. After being placed at 2℃-8℃ and -20℃ for two weeks, the purity of each sample did not change significantly, indicating that the samples have good stability. Table H1: Purity of Formulations 1-12 under different temperature and time conditions (R-CE-SDS)

[0167] According to the NR-CE-SDS purity results in Table H2, after being placed at high temperature for 4 weeks, the purity of all samples decreased to some extent, but the purity decrease trend of formulations 1-12 was not significantly different. Table H2: Purity of Formulations 1-12 under different temperature and time conditions (NR-CE-SDS)

[0168] Analysis of the DAR values ​​of formulations 1-12 showed that under any different storage conditions, there was no significant change compared to the initial value of 3.5, indicating that the formulations have good stability.

[0169] Table I1 shows that the detachment of the linker-loaded material was relatively slow at 2℃-8℃ and -20℃. At 20℃ and 40℃, the detachment was relatively fast, but there was no significant difference in the detachment rate among formulations 1-12. However, the detachment rate was significantly correlated with the pH of the buffer. Lower pH accelerated the detachment, while higher pH slowed it down. Table I1: Linker-loaded content (mg / ml) of formulations 1-12 under different temperature and time conditions

[0170] The data in Table I2 show a similar trend; the free drug content of each formulation increases under conditions of higher temperature and lower pH. Table I2: Free drug content (mg / ml) of formulations 1-12 under different temperature and time conditions

[0171] Table J shows the binding activity test results of formulations 7 to 9 after storage under different conditions, as determined by ELISA. The data indicate that, under all the temperature conditions investigated, the binding activity of the ADC did not change significantly after being stored in a histidine buffer system at a pH range of 5.4 to 6.2 for 4 weeks. Table J: ELISA results of binding activity percentage of formulations 7-9 under different temperature and time conditions. 3.4 Preliminary formulation screening conclusions

[0172] Based on the results of visible foreign matter, insoluble particles, and SEC analysis, it was found that ADCs are more likely to form aggregates and particles in citrate and succinate buffers. Furthermore, SEC results indicated that, under the same buffer system conditions, the higher the pH, the faster the aggregate formation rate.

[0173] CEX test results show that when the pH is below 6.0, the acidic and alkaline components increase rapidly. When the pH is above 6.0, the increase in acidic and alkaline components is slower.

[0174] Free drug analysis showed that the rate of loading detachment was significantly correlated with the pH of the buffer. Lower pH values ​​accelerated loading detachment, while higher pH values ​​slowed it down. Acetate buffers have a relatively low pH range (pH 4.8 to 5.4).

[0175] In summary, based on the results of the first round of formulation screening, a 20mM histidine buffer with a pH range of 5.8±0.3 (i.e., pH 5.5 to 6.1) was selected as the buffer system for the next round of screening experiments. Example 4: Further Formulation Screening 4 4.1 Formation of ADC buffer formulation 13-24

[0176] Twelve further buffer formulations according to Table K were prepared. Dialysis bags were immersed in the appropriate buffer solutions containing stabilizers as shown in Table K. One end was sealed with a dialysis clamp, and the ADC sample (37.4 mg / ml) was carefully transferred into the dialysis bag, with the bag opening clamped. The bag was placed in a beaker containing the buffer solution according to Table K and magnetically stirred at room temperature, with the dialysis solution changed twice during this period. After dialysis at room temperature, the dialysis bag was transferred to fresh dialysis solution and dialyzed overnight at 2°C–8°C. During each dialysis solution change, the volume of liquid replaced did not exceed 1:100. After dialysis, protein concentration and pH were measured, and the sample was diluted with the appropriate buffer to the target concentration of 20.0 mg / ml. Finally, freshly prepared surfactants (10% PS 20 or PS 80) were added to each formulation until the level specified in Table K was reached.

[0177] Preparation 13-24 was aseptically filled into 2R vials (1.0 ml / vial) on a clean bench and then capped and corked. Preparation 13-24 was then stored for up to 3 months under various conditions (-20°C, 2°C-8°C, 25°C, and 40°C) and analyzed periodically as described in Section 4.2.

[0178] Formulations 13-24 were subjected to freeze-thaw cycles (3 or 5 cycles), shaking for up to 5 days, and light exposure for up to 10 days. Freeze-thaw cycles were performed using a -20°C freezer (Haier, DW-25L262). Shaking was conducted at 25°C and 300 rpm using a constant-temperature shaker (Hrystal, IS-RDS3). Light exposure was tested at 4500 ± 500 lux in a light exposure testing chamber (Binder, KBF P 240(E6)). Table K: Summary of Further Formulations 4.2 Analysis of ADC buffer formulations 1-12

[0179] The protein concentration and insoluble particle level of formulation 13-24 were determined according to the analytical methods described in the General Methods section above. Aggregates / monomers / fragments were determined by SEC-HPLC, charge heterogeneity was determined by CEX-HPLC, purity, DAR, and free drug level were determined by R-CE-SDS / NR-CE-SDS, and binding activity was determined by ELISA.

[0180] The osmotic pressure of the samples was measured using an Osmomat 3000 osmometer. Calibration was performed using calibration solutions (300 mOsm / L, 500 mOsm / L) before sample testing. 4.3 Preliminary formulation screening results

[0181] The protein concentration, pH, and osmotic pressure of the buffer formulations after various treatments (where F / T = freeze-thaw; S = shaking; I = light exposure) are shown in Tables L, M, and N, respectively. Except for the sample exposed to light for 10 days, which showed an increased protein concentration and a decreased pH, these parameters did not show significant changes after the different treatments. Table L: Protein concentrations (mg / ml) of formulations 13 to 24 (ND = not determined) Table M: ​​pH values ​​of formulations 13 to 24 (ND = not determined) Table N: Osmolarity (mOsm / L) of Formulations 13 to 24 (ND = Not determined)

[0182] Regarding the appearance of the formulation, no obvious visible foreign matter was observed in any sample at T=0 or after the above treatment. Except for the sample that turned yellowish-brown after 10 days of light exposure, all samples were colorless, transparent or slightly milky liquids.

[0183] As shown in Tables O1-O3, formulations 13-24 exhibited relatively low levels of insoluble particles at different time points under various testing conditions. As shown in Table O1, formulation 14 performed slightly better than formulation 13 in terms of particle levels at T=0 and after repeated freeze-thaw cycles and light exposure. In terms of freeze-thaw cycles and oscillation, formulation 14 performed slightly better than formulation 16. The only difference between formulations 14 and 13 is the surfactant component (0.03% polysorbate 80 in formulation 14; 0.03% polysorbate 20 in formulation 13), and the only difference between formulations 14 and 16 is the stabilizer component (6% sucrose in formulation 14; 6% trehalose in formulation 16). Polysorbate 80 and sucrose appear to inhibit particle formation in ADC formulations. Table O1: Insoluble particle levels after different treatments (≥2μm; ≥5μm; ≥10μm; ≥25μm; unit = particles / ml) Table O2: Insoluble particle levels under different temperature and time conditions (A: ≥2μm; B: ≥5μm; Unit: particles / ml); (Indicates that no valid data was obtained) Table O3: Insoluble particle levels under different temperature and time conditions (C: ≥10μm; D: ≥25μm; Unit: particles / ml; (Indicates that no valid data was obtained)

[0184] Under different conditions, the proportions of aggregates, monomers, and fragments in all formulations did not differ significantly. After 4 weeks of storage at 40°C, the proportion of aggregates increased by 0.9%–1.2%, while the proportion of monomers decreased by 1.7%–2.0%. However, after exposure to light, the proportion of aggregates increased significantly, while the proportion of monomers decreased significantly. SEC-HPLC results are shown in Tables P1 to P3. Table P1: Percentage of aggregates in formulations 13-24 under different conditions (SEC-HPLC) Table P2: Percentage content of monomers in formulations 13-24 under different conditions (SEC-HPLC) Table P3: Fragment percentage content of formulations 13-24 under different conditions (SEC-HPLC)

[0185] The results of charge heterogeneity (CEX-HPLC) are shown in Tables Q1-Q3. After 4 weeks of storage at 40°C, (i) the proportion of the main peak in the formulation at pH 5.5 (formulations 17-20) decreased by approximately 12%, while the proportions of acidic and basic components increased by approximately 3.5% and 8.5%, respectively; (ii) the proportion of the main peak in the formulation at pH 5.8 (formulations 13-16) decreased by approximately 10%, while the proportions of acidic and basic components increased by approximately 3% and 7%, respectively; and (iii) the proportion of the main peak in the formulation at pH 6.1 (formulations 21-24) decreased by approximately 8%, while the proportions of acidic and basic components increased by approximately 3% and 5%, respectively. This indicates that the ADC molecule is more stable in the formulation at pH 6.1. After 3 months of observation at 25°C, the main peak also showed a certain degree of decrease, and the proportions of acidic and basic components increased slightly. After 5 and 10 days of light exposure, the CEX spectra changed significantly (making charge isomer analysis impossible), indicating that the samples had been significantly damaged. Under other conditions, the charge isomers of each system did not change significantly. Table Q1: Acid peak content of formulations 13-24 under different conditions (determined by CEX-HPLC) Table Q2: Peak content of formulations 13-24 under different conditions (determined by CEX-HPLC) Table Q3: Basic peak content of formulations 13-24 under different conditions (determined by CEX-HPLC)

[0186] Purity (NR CE-SDS) results are shown in Table R. Sample purity decreased significantly after 5 days of light exposure. After 4 weeks at 40°C, sample purity decreased slightly, but the decreasing trend was not significantly different among the 12 formulation samples. Under other testing conditions, sample purity did not change significantly. Table R: Purity of Formulations 13-24 under different conditions (NR-CE-SDS)

[0187] Analysis of the DAR values ​​of formulations 13-24 showed that under any different storage conditions, there was no significant change compared to the initial value of 3.5 (except for the significant change in the HIC spectrum after 5-10 days of light exposure, which made DAR value analysis impossible), indicating that the samples had undergone significant degradation.

[0188] Table S1 shows that the detachment of the linker-load was relatively slow at 2℃-8℃ and -20℃, and after freeze-thaw cycles. At 25℃ and 40℃, the detachment was relatively fast, but there was no significant difference in the detachment rate among formulations 13-24, indicating that the different pH and buffer systems investigated had no effect on detachment. Table S1: Linker-Load Content (mg / ml) of Formulations 13-24 under Different Conditions

[0189] Table S2 shows that the detachment of the loading (free drug) was relatively slow under freeze-thaw and shaking conditions at 2℃-8℃ and -20℃, but detachment was faster at 25℃ and 40℃. At 40℃ storage conditions, the concentration of the loading increased significantly faster in samples with pH 5.5 (formulations 17-20) and pH 5.8 (formulations 13-16) compared to formulations with pH 6.1 (formulations 21-24). This demonstrates that the detachment of the loading from the ADC is slower at pH 6.1.

[0190] After 5 and 10 days of light exposure, a large number of unknown impurity peaks appeared in the free drug detection spectrum, indicating that the sample had undergone significant degradation. Table S2: Free drug content (mg / ml) of formulations 13-24 under different conditions

[0191] Regarding binding activity measured by ELISA, the relative binding activity of the tested samples decreased significantly (both below 60%) after 5 and 10 days of light exposure, indicating significant sample degradation. Under other testing conditions, the relative binding activity of different formulation samples did not change significantly. 4.4 Conclusions of further formulation screening

[0192] Based on visible foreign matter, SEC purity, non-reduced CE-SDS purity, DAR value, and relative binding activity, the results showed no significant differences among the 12 different formulations. Insoluble particle analysis showed that the surfactant polysorbate 80 performed slightly better than polysorbate 20, and the stabilizer sucrose performed slightly better than trehalose. Both CEX analysis and free drug analysis showed that the buffer system at pH 6.1 performed better than buffer systems at pH 5.5 or pH 5.8.

[0193] Therefore, further formulation screening identified formulation 23 as the preferred ADC formulation, which comprises: i) 20mg / ml ADC ( Active ingredients ); ii) 20 mM histidine / histidine hydrochloride, pH 6.1 buffer ); iii) 6% sucrose (W:V) stabilizer );and iv) 0.03% Polysorbate 80 (W:V) surfactants ).

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate (ADC) and a buffer, wherein the ADC has the following structure: Where X is and A mixture; and A is the anti-TROP2 antibody GQhRS7 or a fragment thereof; The linker and the antibody or antibody fragment are linked to at least one light chain of the antibody or antibody fragment via an amide bond; The pharmaceutical composition is a liquid and has a pH of 5.4 to 6.

5.

2. The pharmaceutical composition of claim 1, wherein the linker and the antibody or antibody fragment are linked by an amide bond at the C-terminus of at least one light chain of the antibody or antibody fragment.

3. The pharmaceutical composition of claim 1, wherein the linker and the antibody or antibody fragment are linked by an amide bond at the C-terminus of each light chain of the antibody or antibody fragment.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the ADC has a mean drug-antibody ratio (DAR) in the range of 2 to 4, preferably 3.

5.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is an aqueous liquid composition.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is in the form of an injectable aqueous solution.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is in the form of an injectable aqueous solution for administration by intravenous infusion.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the ADC is dissolved in the pharmaceutical composition at a concentration of 5 mg / mL to 100 mg / mL, preferably 5 mg / mL to 50 mg / mL, more preferably 15 mg / mL to 25 mg / mL, and even more preferably 20 mg / mL, wherein the concentration refers to the protein concentration of the ADC dissolved in the pharmaceutical composition.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition has a pH of 5.5 to 6.2, preferably 5.8 to 6.2, and more preferably 6.0 to 6.

2.

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition has a pH of 6.

1.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the buffer is a histidine buffer.

12. The pharmaceutical composition of claim 11, wherein the histidine buffer comprises a mixture of L-histidine and L-histidine monohydrochloride.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the buffer component is present at a concentration of 5 mM to 50 mM, preferably 10 mM to 30 mM, more preferably 20 mM.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition further comprises a surfactant.

15. The pharmaceutical composition according to claim 14, wherein the surfactant is polysorbate, preferably polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

16. The pharmaceutical composition of claim 14 or 15, wherein the pharmaceutical composition comprises 0.01% to 0.2% w / v, preferably 0.01% to 0.1% w / v, more preferably 0.02% to 0.05% w / v, and even more preferably 0.03% w / v of the surfactant.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the pharmaceutical composition further comprises a stabilizer.

18. The pharmaceutical composition according to claim 17, wherein the stabilizer is a sugar stabilizer selected from the group consisting of: trehalose, mannitol, sucrose, maltose, lactose, fructose, xylitol, arabinitol, erythritol, sylitol, sorbitol, raffinose, lactitol, maltitol, and inositol.

19. The pharmaceutical composition according to claim 17, wherein the stabilizer is a non-reducing sugar, preferably selected from sucrose or trehalose, and more preferably sucrose.

20. The pharmaceutical composition according to any one of claims 17 to 19, wherein the pharmaceutical composition comprises 0.5% to 10% w / v, preferably 3% to 8% w / v, more preferably 4% to 8% w / v, and even more preferably 6% w / v of a stabilizer.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, such as tension modifiers, antioxidants and / or diluents.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the molar osmotic pressure concentration of the pharmaceutical composition is from 150 mOSm / kg to 450 mOSm / kg, preferably from 200 mOSm / kg to 400 mOSm / kg.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the ADC maintains physical and / or chemical stability within the pharmaceutical composition for at least four weeks when stored at 2°C-8°C or -20°C.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the ADC is stable within the pharmaceutical composition and has less than 5% aggregates as determined by SEC-HPLC when stored at 2°C-8°C or -20°C for at least four weeks.

25. The pharmaceutical composition according to any one of claims 1 to 24, wherein the pharmaceutical composition is in the form of an injectable aqueous solution for administration by intravenous infusion, and the solution is substantially or completely free of particulate matter.

26. A method for preparing a pharmaceutical composition according to any one of claims 1 to 25, the method comprising the following steps: The ADC and the buffer, along with any optional one or more other components, may be mixed together in any amount, concentration, or form; and any one or more parameters relating to the pharmaceutical composition, such as pH, may be adjusted.

27. A product obtained by or from a method of preparing a pharmaceutical composition as defined in claim 26.

28. A lyophilized formulation comprising an ADC and a buffer, wherein the formulation, upon reconstitution, is capable of forming the pharmaceutical composition of any one of claims 1 to 25.

29. A method for preparing a lyophilized formulation, wherein the method comprises lyophilizing the pharmaceutical composition of any one of claims 1 to 25.

30. A container containing a pharmaceutical composition of any one of claims 1 to 25 or a lyophilized formulation of claim 28, wherein the container is a vial, a disposable vial, a light-protected vial, an ampoule, a syringe, a pre-filled syringe, an injection pen, or an intravenous infusion bag.

31. The pharmaceutical composition of any one of claims 1 to 25 or the lyophilized formulation of claim 28 is used as a medicine.

32. A method of treating a subject with cancer or an autoimmune disease, the method comprising administering a therapeutically effective amount of any one of claims 1 to 25 or the lyophilized formulation of claim 28.

33. A method of treating cancer in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 25 or the lyophilized formulation of claim 28.

34. The method of claim 33, wherein the cancer is a TROP2-associated tumor.

35. The method of claim 34, wherein the TROP2-related tumor comprises a tumor that overexpresses TROP2 or a tumor containing one or more TROP2 gene mutations.

36. The method according to any one of claims 30 to 35, wherein the cancer is selected from fibrosarcoma, myoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, and sweat gland cancer. Cancer, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

37. The method according to any one of claims 33 to 36, wherein the cancer is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

38. The method according to claim 32 or 37, wherein the subject is a mammal, preferably a human.

39. A combination kit comprising a pharmaceutical composition of any one of claims 1 to 25 or a lyophilized formulation of claim 28 in a container, and optionally a set of instructions for use, the instructions having guidance on the administration of the pharmaceutical composition or the lyophilized formulation (e.g., by intravenous infusion).

40. A combination kit comprising, in a container, the lyophilized formulation of claim 28, a diluent for reconstituted with the lyophilized formulation, and optionally a set of instructions for administration of the reconstituted formulation (e.g., by intravenous infusion).

Images