Pharmaceutical composition of antibody-drug conjugate

Abstract

Provided are a pharmaceutical composition of an antibody-drug conjugate, and a preparation method therefor and a use thereof.

Description

A pharmaceutical composition of an antibody-drug conjugate

[0001] Citation of relevant applications

[0002] This application claims priority to Chinese Patent Application No. 202411506696.5, filed on October 28, 2024, the entire contents of which are incorporated herein by reference and used for all purposes. Technical Field

[0003] This application relates to a pharmaceutical composition of an antibody-drug conjugate, as well as a method for preparing the composition and its use. Background Technology

[0004] Antibody-drug conjugates (ADCs) consist of three distinct components: antibody, linker, and drug. ADC technology uses a linker to conjugate antibody and drug molecules together, utilizing the antibody's specific targeting to deliver drug molecules to the target tissue to exert their effects, reducing systemic toxicity of drugs, expanding the therapeutic window of drugs, and enhancing the therapeutic potential of antibodies.

[0005] Folic acid receptor α (FRα) is a cell surface glycoprotein with a molecular weight of 38-40 kDa, encoded by the FOLR1 gene. Its main function is to transport folic acid, but its expression level is significantly increased in rapidly proliferating cancer cells, participating in the regulation of tumor proliferation and metastasis. Therefore, it is also one of the targets for antibody or ADC drug development.

[0006] In the study of antibody and antibody-drug conjugate (ADC) formulations, the formation of aggregates and the generation of degradation products can cause pharmaceutically undesirable side effects, leading to increased immunogenicity or intravenous disease-related risks in patients receiving drug treatment. For these reasons, it is necessary to inhibit aggregate formation and degradation product generation when formulating related formulations. Particularly when studying drug formulations of antibody-drug conjugates, more technical challenges arise, requiring consideration not only of the specific properties of the antibody moiety but also of the drug-connector moiety. For example, small molecule toxins may detach during storage of antibody-drug conjugates, which can affect the efficacy and toxicity of the related drugs.

[0007] Developing stable drug compositions and addressing the aggregation and degradation issues of ADC molecules in antibody-drug conjugate (ADC) compositions are among the research directions in the ADC field.

[0008] Invention Overview

[0009] In a first aspect, this application provides a pharmaceutical composition comprising an antibody-drug conjugate of Formula I:

[0010] Where A is an antibody or its antigen-binding fragment targeting folate receptor α (FRα); R is selected from... Wherein, Ra is selected from C1-C3 alkylene groups, Rb is selected from C1-C3 alkyl groups, r, t, and u are each independently selected from integers in the range of 1-50, and m is an integer or decimal in the range of 1 to 12.

[0011] The pharmaceutical composition is a liquid preparation, a lyophilized preparation, or a powder for injection.

[0012] Secondly, this application provides a method for preparing the pharmaceutical composition described in the first aspect, comprising the following steps:

[0013] (1) Preparation of the antibody-drug conjugate shown in Formula I;

[0014] (2) Preparation of ultrafiltration replacement buffer; and

[0015] (3) Change the antibody-drug conjugate prepared in step (1) to the buffer solution prepared in step (2).

[0016] Thirdly, this application provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for treating proliferative diseases.

[0017] Fourthly, this application provides a method for treating or preventing proliferative diseases, the method comprising administering a therapeutically effective dose of the pharmaceutical composition described in the first aspect to a patient in need of it. Attached Figure Description

[0018] Figures 1A and 1B show the single-crystal diffraction patterns of compound 82.

[0019] Figure 2 shows the SEC-HPLC detection results of the exemplary ADC of this application after high temperature and light treatment at different pH and buffer salts.

[0020] Detailed description of the invention

[0021] Terminology Definition

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0023] Unless otherwise specified, the term "antibody-drug conjugate (ADC)" refers to the linking of an antibody (such as a monoclonal antibody) or antibody fragment to a cytotoxic drug having biological activity (e.g., therapeutic activity, more specifically, anticancer activity) via a stable chemical linker compound.

[0024] Unless otherwise specified, the term "linker-drug compound" refers to the partial structure of an "antibody-drug conjugate" consisting of a linker compound and a drug compound, and is also referred to in this application as "linker-drug" or the abbreviation "LD".

[0025] This application names exemplary ADC molecules using the format of antibody code-ADC linker-drug compound code-expected DAR. For example, in "F-ADC1-1-8", "F" indicates that the ADC molecule contains Farletuzumab as the antibody portion, LD1-1 as the linker-drug compound, and the expected conjugation DAR is 8. The naming meanings of other exemplary ADC molecules follow the same logic. Those skilled in the art will understand that because the actual conjugation efficiency of the antibody and LD molecule may not reach the designed 100%, the measured / actual DAR may differ from the expected conjugation DAR for the ADC molecule population or ADC formulation. For example, the measured / actual DAR of "F-ADC1-1-8" shown in Table 2 is 7.6, which is for the ADC sample and does not contradict the expected DAR. Based on the relevant results, those skilled in the art can also determine that the "F-ADC1-1-8" sample tested in Table 2 mainly contains ADC molecules with a DAR of 8.

[0026] The linker-drug compound described in this application is linked to the antibody via conventional coupling methods in the art, including: lysine coupling, inter-chain reducing disulfide bond coupling, and directional coupling (Beck, Alain, and Janice M. Reichert. "Antibody-drug conjugates: present and future." MAbs. Vol. 6. No. 1. Taylor & Francis, 2014.; McCombs, Jessica R., and Shawn C. Owen. "Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry." The AAPS journal 17(2015): 339-351.). This application preferably uses inter-chain reducing disulfide bond coupling, i.e., the linkage is achieved by the reaction of one or more thiol groups (sulfur atoms of cysteine ​​residues) formed after reduction at the disulfide bond sites between the light and heavy chains (two sites between the heavy chain and two sites between the heavy and light chains).

[0027] Unless otherwise specified, the term "about" means a value within an acceptable margin of error for a specific value determined by a person skilled in the art, the value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, each occurrence of "about" in the art may mean a range of ±20%, ±15%, ±10%, ±5%, ±1% for the specific value subsequently indicated.

[0028] All ranges disclosed herein should be understood to encompass any and all subranges contained herein. For example, the range “1 to 10” described herein should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (inclusive); that is, all subranges that begin with the minimum value 1 or greater, such as 1 to 6.1, and subranges that terminate with the maximum value 10 or less, such as 5.5 to 10.

[0029] The term “object” or “individual” as used in this article refers to mammals, such as humans, but can also refer to other animals, such as wild animals, livestock, or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0030] In a broad sense, an "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Therefore, it encompasses complete antibodies / full-length antibodies, single-chain antibodies, or any antigen-binding fragment of an antibody (also known as an "antigen-binding moiety"). When "antibody" and "antigen-binding fragment / antigen-binding moiety" appear in the same context, "antibody" can be understood as the complete entity relative to the "antigen-binding fragment / antigen-binding moiety," and both correspond to the broad concept of antibody.

[0031] A "full-length antibody" refers to a protein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into multiple highly variable regions called complementarity-determining regions (CDRs), interspersed with multiple more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be designated into different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Typically, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chain corresponding to different immunoglobulin classes are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also included in the antibodies according to this application. It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of an antibody. There are several common ways to define the CDR amino acid sequence of VH or VL, such as the Kabat definition, the IMGT definition, and the Chothia definition. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can usually be determined according to different definitions. In the embodiments of this application, the Kabat definition of the CDR amino acid sequence is used. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed using various methods.

[0032] The term "monoclonal antibody" refers to an antibody obtained from a population of essentially homogeneous antibodies, meaning that the individual antibodies that make up the population are identical, except that naturally occurring mutations may exist in a small number of individuals.

[0033] As used herein, the terms “antigen-binding fragment” or “antigen-binding portion” or “antigen-binding region” are used interchangeably to refer to a portion of an antibody containing amino acid residues that interact with an antigen and confer binding specificity and affinity against that specific antigen, particularly antibody fragments such as Fv, Fab, F(ab')2, or Fab', or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols such as polyethylene glycol (“PEGylation”) (a PEGylation fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) (“PEG” stands for polyethylene glycol), which has FRα binding activity. Preferably, the antigen-binding fragment will consist of or contain a portion of the heavy or light chain variable chain of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, and such antigen-binding fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of its source antibody sequence. Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) Fv fragments having a single arm of antibody with VL and VH domains; and (4) VHH fragments consisting of VH domains.

[0034] As used herein, the term "pharmaceutical composition" means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient, which are combined together to achieve a particular purpose.

[0035] In this application, the term "effective amount" means an amount that has a therapeutic effect on a subject, such as: in subjects who have been given the effective amount, the symptoms or state of the disease are alleviated, reduced, or eliminated, or the development of the symptoms or state of the disease is delayed or suppressed, compared to subjects who have not been given the effective amount.

[0036] As used in this article, the term "DAR" refers to the ratio of drug (D) to antibody (A) in an antibody-drug conjugate, or the ratio of linker-drug compound (LD) to antibody (A). For example, a DAR value of 8 means that one antibody molecule is conjugated to 8 linker-drug compounds. When the DAR value is a decimal, it refers to the average number of antibody-drug conjugates with linker-drug compounds (LD) in the antibody-drug conjugate population.

[0037] In a first aspect, this application provides a pharmaceutical composition comprising an antibody-drug conjugate of Formula I:

[0038] Wherein, A is an antibody or its antigen-binding fragment targeting folate receptor α (FRα), and R is selected from... Wherein, Ra is selected from C1-C3 alkylene (e.g., methylene, ethylene, n-propylene, or isopropylene); Rb is selected from C1-C3 alkyl (e.g., methyl, ethyl, n-propyl, or isopropyl); r, t, and u are each independently selected from integers in the range of 1-50 (e.g., 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50); m is an integer or decimal in the range of 1 to 12; and

[0039] The pharmaceutical composition is a liquid preparation, a lyophilized preparation, or a powder for injection.

[0040] In some implementations, r, t, and u are each independently selected from integers in the range of 4 to 30.

[0041] In some implementations, m is selected from an integer in the range of 4-8 or a decimal in the range of 4-8.

[0042] In some implementations, when m is an integer selected from the range 1-12, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0043] In some implementations, when m is a decimal, it represents the average number of linker-drug molecules conjugated to each antibody unit in the drug composition and the ADC molecule.

[0044] In some embodiments, the antibody-drug conjugate represented by Formula I has the following structure:

[0045] In some embodiments, the antibody-drug conjugate represented by Formula I has the following structure:

[0046] In some embodiments, the antibody or antigen-binding fragment of the FRα represented by A comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementarity-determining regions (CDRs), wherein the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:3; and the light chain comprises three complementarity-determining regions (CDRs), wherein the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID NO:6, wherein the CDRs are determined according to the Kabat numbering rules.

[0047] In some embodiments, the antibody or antigen-binding fragment of the A-representing FRα-targeting region includes a heavy chain variable region and / or a light chain variable region. The heavy chain variable region includes three complementarity-determining regions (CDRs), wherein the amino acid sequence of heavy chain CDR1 (HCDR1) is shown in SEQ ID NO:1, the amino acid sequence of heavy chain CDR2 (HCDR2) is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain CDR3 (HCDR3) is shown in SEQ ID NO:3; the light chain variable region includes three complementarity-determining regions (CDRs), wherein the amino acid sequence of light chain CDR1 (LCDR1) is shown in SEQ ID NO:4, the amino acid sequence of light chain CDR2 (LCDR2) is shown in SEQ ID NO:5, and the amino acid sequence of light chain CDR3 (LCDR3) is shown in SEQ ID NO:6, wherein the CDRs are determined according to the Kabat numbering rules. In some embodiments, the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region (VL) is shown in SEQ ID NO:8.

[0048] In some embodiments, the antibody targeting FRα or its antigen-binding fragment comprises a heavy chain variable region (VH) with an amino acid sequence as shown in SEQ ID NO:7 and a light chain variable region (VL) with an amino acid sequence as shown in SEQ ID NO:8.

[0049] In some implementations, antibodies targeting FRα also include a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0050] In some implementations, the antigen-binding fragment of the antibody targeting FRα is selected from Fab, Fab', Fab'-SH, Fv, scFv, or F(ab')2.

[0051] In some implementations, the antibody targeting FRα or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0052] In some embodiments, the antibody targeting FRα comprises a heavy chain and / or a light chain, the amino acid sequence of which is shown in SEQ ID NO:9 or SEQ ID NO:11, and the amino acid sequence of which is shown in SEQ ID NO:10.

[0053] In this application, the CDR amino acid sequence numbering rule adopts the Kabat numbering rule.

[0054] In some implementations, the antibody targeting FRα is Farletuzumab (also abbreviated as "F" herein), which comprises two identical heavy and light chains (WO 2023 / 170247 A1), with the heavy chain amino acid sequence shown in SEQ ID NO:9 and the light chain amino acid sequence shown in SEQ ID NO:10.

[0055] Farletuzumab heavy chain amino acid sequence (SEQ ID NO:9):

[0056] Farletuzumab light chain amino acid sequence (SEQ ID NO:10):

[0057] The amino acid sequences indicated in bold are the heavy chain variable region / light chain variable region CDR amino acid sequences. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0058] The heavy chain variable region amino acid sequence VH and the light chain variable region amino acid sequence VL of Farletuzumab are SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0059] Farletuzumab heavy chain variable region (VH) sequence (SEQ ID NO:7):

[0060] Farletuzumab light chain variable region (VL) sequence (SEQ ID NO:8):

[0061] In some implementations, the antibody targeting FRα is Farletuzumab-FcS (also abbreviated as "FS" herein), which comprises two identical heavy and light chains, the heavy chain amino acid sequence of which is shown in SEQ ID NO:11 and the light chain amino acid sequence of which is shown in SEQ ID NO:10.

[0062] Farletuzumab-FcS heavy chain amino acid sequence (SEQ ID NO:11):

[0063] Farletuzumab-FcS light chain amino acid sequence (SEQ ID NO:10):

[0064] The amino acid sequences indicated in bold are heavy chain / light chain CDR amino acid sequences. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0065] The amino acid sequences of the heavy chain variable region sequence VH and the light chain variable region sequence VL of Farletuzumab-FcS are SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0066] Farletuzumab-FcS heavy chain variable region (VH) amino acid sequence (SEQ ID NO:7):

[0067] Farletuzumab-FcS light chain variable region (VL) amino acid sequence (SEQ ID NO:8):

[0068] In some embodiments, the antibody-drug conjugates represented by Formula I have the structures shown in Table 1 (the left column indicates the molecule numbers in this application):

[0069] Table 1

[0070] Where m is an integer or decimal in the range of 2-8, preferably selected from 2, 4, 6 or 8.

[0071] In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation. In some embodiments, the pharmaceutical composition is an injectable formulation. In some embodiments, the pharmaceutical composition is an injection solution or a lyophilized injection. In some embodiments, the pharmaceutical composition is a lyophilized injection.

[0072] Lyophilized injections are solid injections (also known as lyophilized powders) that can be used by dissolving them in a solvent (preferably water, more preferably water for injection) when in use. They are obtained by lyophilizing a stock solution containing a predetermined amount of the pharmaceutical components in a solvent (preferably ethanol, methanol, or water).

[0073] In this application, water for injection refers to water that meets the requirements of the Chinese Pharmacopoeia (2020) under the category of water for injection.

[0074] In some embodiments, the pharmaceutical composition further comprises a buffer.

[0075] In some embodiments, the pharmaceutical composition further comprises a stabilizer.

[0076] In some embodiments, the pharmaceutical composition further comprises a surfactant.

[0077] In some embodiments, the pharmaceutical composition further comprises any two or all three of a buffer, a stabilizer, and a surfactant.

[0078] In some embodiments, the pharmaceutical composition further comprises water (for lyophilized formulations, this refers to residual moisture from the lyophilization process).

[0079] In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate of Formula I, a buffer, a stabilizer, and a surfactant.

[0080] In some embodiments, the antibody-drug conjugate represented by Formula I is selected from the ADC molecules shown in Table 1, such as the exemplary ADC molecules of this application: F-ADC-1, F-ADC-1-1, F-ADC-1-2, F-ADC-2, F-ADC-2-1, F-ADC-2-2, F-ADC-3, F-ADC-3-1, F-ADC-3-2, FS-ADC-1, FS-ADC-1-1, FS-ADC-1-2, FS-ADC-2, FS-ADC-2-1, FS-ADC-2-2, FS-ADC-3, FS-ADC-3-1 or FS-ADC-3-2.

[0081] In some embodiments, the buffer is selected from one or more of acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, tromethamine (Tris), and morpholine ethanesulfonic acid (MES). Other organic acid buffers suitable for ADC formulations are also applicable to this application.

[0082] In some embodiments, the histidine buffer is a buffer containing histidine ions. Examples of histidine buffers are selected from histidine-histidine hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., wherein the histidine-histidine hydrochloride buffer is prepared by reacting histidine with histidine hydrochloride, and the histidine-acetate buffer is prepared by reacting histidine with acetic acid. Similarly, the succinate buffer can be succinic acid-sodium succinate, and the citrate buffer can be citric acid-sodium citrate.

[0083] In some implementations, the buffer is a histidine-histidine hydrochloride buffer.

[0084] In some embodiments, the stabilizer is selected from: sugars (such as sucrose and / or trehalose), polyols (such as mannitol and / or sorbitol), and amino acids (such as L-serine, monosodium glutamate, alanine, glycine, or sarcosine).

[0085] In some embodiments, the surfactant is selected from polysorbates, such as polysorbate 20 and / or polysorbate 80.

[0086] In the embodiments described below regarding pH, mass percentage (in mass %), and content (in mass / volume, such as mg / ml), unless otherwise specified, the pH value of the pharmaceutical composition is the pH value of an aqueous solution of the pharmaceutical composition (wherein the water is water for injection); unless otherwise specified, when describing mass percentage, it is based on a total weight of the pharmaceutical composition of 100 wt%, and is premised on the pharmaceutical composition excluding water for injection; unless otherwise specified, when describing content, the pharmaceutical composition includes water for injection and is based on the volume of the pharmaceutical composition.

[0087] In some embodiments, the pH of the pharmaceutical composition is 4.0-7.5 (e.g., about 4.0, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.5, about 7.0, or about 7.5). In some embodiments, the pH of the pharmaceutical composition is 4.5-7.0. In some embodiments, the pH of the pharmaceutical composition is 4.5-5.5. In some embodiments, the pH of the pharmaceutical composition is 5.0-5.5. In some embodiments, the pH of the pharmaceutical composition is about 5.3.

[0088] In the above implementation scheme and similar implementation schemes below, the "about" before the point value includes the value itself as well as various values ​​within the error range that can be understood by those skilled in the art.

[0089] In some embodiments, the pharmaceutical composition is a lyophilized injection, optionally containing residual moisture.

[0090] In some embodiments, the pharmaceutical composition contains 1-30% by mass of the antibody-drug conjugate of Formula I (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%). In some embodiments, the pharmaceutical composition contains 5-25% by mass of the antibody-drug conjugate of Formula I. In some embodiments, the antibody-drug conjugate of Formula I in the pharmaceutical composition comprises 10-25% by mass. In some embodiments, the antibody-drug conjugate of Formula I in the pharmaceutical composition comprises about 19% by mass.

[0091] In some embodiments, the buffer (such as histidine-histidine hydrochloride) in the pharmaceutical composition comprises 1-10% by mass (e.g., about 1%, about 2%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, or about 10%). In some embodiments, the buffer (such as histidine-histidine hydrochloride) in the pharmaceutical composition comprises 1-9.9% by mass. In some embodiments, the buffer (such as histidine-histidine hydrochloride) in the pharmaceutical composition comprises 2.5-7.5% by mass. In some embodiments, the buffer (such as histidine-histidine hydrochloride) in the pharmaceutical composition comprises about 5% by mass.

[0092] In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine content is 0.01-2% by mass (e.g., about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, or about 2%). In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine content is 0.25-0.75% by mass. In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine content is about 0.5% by mass.

[0093] In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine hydrochloride is present in a mass percentage of 1-10% (e.g., about 1%, about 2%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.5%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, or about 10%). In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine hydrochloride is present in a mass percentage of 1-9.9%. In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine hydrochloride comprises 2-7% by mass. In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine hydrochloride comprises about 4.5% by mass.

[0094] In some embodiments, the stabilizer (such as trehalose or sucrose) in the pharmaceutical composition comprises 1-90% by mass (e.g., about 1%, about 5%, about 6%, about 7%, about 8%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, or about 90%). In some embodiments, the stabilizer (such as trehalose or sucrose) in the pharmaceutical composition comprises 50%-90% by mass. In some embodiments, the stabilizer (such as trehalose or sucrose) in the pharmaceutical composition comprises about 75.7% by mass.

[0095] In some embodiments, the surfactant (such as polysorbate 80) in the pharmaceutical composition is present in a mass percentage of 0.01-1% (e.g., about 0.01%, about 0.02%, about 0.05%, about 0.1%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%). In some embodiments, the surfactant (such as polysorbate 80) in the pharmaceutical composition comprises 0.05-0.4% by mass. In some embodiments, the surfactant (such as polysorbate 80) in the pharmaceutical composition comprises 0.3% by mass.

[0096] In some embodiments, the pharmaceutical composition comprises, by weight percentage:

[0097] The antibody-drug conjugates shown in Formula I (e.g., any of the ADC molecules shown in Table 1) have a mass percentage of 1-30%, 5-25%, 10-25%, or about 19%.

[0098] Buffers (such as histidine-histidine hydrochloride buffers), with a mass percentage of 1-10%, 1-9.9%, 2.5-7.5%, or approximately 5%.

[0099] Stabilizers (such as trehalose and / or sucrose) at a mass percentage of 1-90% or 50%-90% or about 75.7%;

[0100] The surfactant (such as polysorbate 80) has a mass percentage of 0.01-1%, 0.05-0.4%, or about 0.3%.

[0101] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, wherein the mass percentage of histidine is 0.1-1% or 0.25-0.75% or 0.5%, and the mass percentage of histidine hydrochloride is 1-10% or 1-9.9% or 2-7% or 4.5%.

[0102] In some embodiments, the pharmaceutical composition comprises, by weight percentage:

[0103] The antibody-drug conjugate shown in Formula I (e.g., any of the ADC molecules shown in Table 1) has a mass percentage of approximately 19%.

[0104] Buffers (such as histidine-histidine hydrochloride buffers) with a mass percentage of approximately 5%;

[0105] Stabilizers (such as trehalose and / or sucrose) at a mass percentage of approximately 75.7%;

[0106] Surfactants (such as polysorbate 80) are present in a mass percentage of approximately 0.3%.

[0107] In some embodiments, the buffer is histidine-histidine hydrochloride, wherein the mass percentage of histidine is about 0.5% and the mass percentage of histidine hydrochloride is about 4.5%.

[0108] In some embodiments, for pharmaceutical compositions in liquid formulation form (e.g., direct injection solutions, solutions for reconstitution of lyophilized formulations, stock solutions used in the preparation of lyophilized formulations), the content of the antibody-drug conjugate shown in Formula I is 1-30 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 10 mg / ml, about 10 mg / ml, etc.). The concentrations of the antibody-drug conjugate shown in Formula I are approximately 5-25 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the concentration of the antibody-drug conjugate shown in Formula I is 10-20 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the concentration of the antibody-drug conjugate shown in Formula I is approximately 15 mg / ml.

[0109] In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the buffer (such as histidine-histidine hydrochloride buffer) is 1-10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml, about 5 mg / ml, about 5.1 mg / ml, about 5.2 mg / ml, about 5.3 mg / ml, about 5.4 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml). In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the buffer (such as histidine-histidine hydrochloride buffer) is 2-6 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the buffer (such as histidine-histidine hydrochloride buffer) is about 4.04 mg / ml.

[0110] In some embodiments of the pharmaceutical composition in liquid formulation form, the buffer is histidine-histidine hydrochloride, wherein the histidine content is 0.1-1 mg / ml (e.g., about 0.1 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml). The concentrations are approximately 0.29 mg / ml, 0.3 mg / ml, 0.31 mg / ml, 0.32 mg / ml, 0.33 mg / ml, 0.34 mg / ml, 0.35 mg / ml, 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml, 0.55 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, or 1 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine content is 0.1-0.5 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine content is 0.2-0.5 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine content is approximately 0.44 mg / ml.

[0111] In some embodiments of the pharmaceutical composition in liquid formulation form, the buffer is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is 1-10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 2.6 mg / ml, about 2.7 mg / ml, about 2.8 mg / ml, about 2.9 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 3.6 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml, about 5 mg / ml, about 5.1 mg / ml, about 5.2 mg / ml, about 5.3 mg / ml, about 5.4 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml). In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is 2-6 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is 2-4 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is about 3.6 mg / ml.

[0112] In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the stabilizer (such as trehalose and / or sucrose) is 30-110 mg / ml (e.g., about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 81 mg / ml, about 82 mg / ml, about 83 mg / ml, about 84 mg / ml, about 85 mg / ml, about 86 mg / ml, about 87 mg / ml, about 88 mg / ml, about 89 mg / ml, about 90 mg / ml, about 91 mg / ml, about 92 mg / ml, about 93 mg / ml, about 94 mg / ml, about 95 mg / ml, about 100 mg / ml, about 105 mg / ml, or about 110 mg / ml). In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the stabilizer (such as trehalose and / or sucrose) is 40-90 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the stabilizer (such as trehalose and / or sucrose) is 40-80 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the stabilizer (such as trehalose and / or sucrose) is about 60 mg / ml.

[0113] In some embodiments of the pharmaceutical composition in liquid formulation form, the content of surfactant (such as polysorbate 80) is 0.005-1 mg / ml (e.g., about 0.005 mg / ml, about 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.25 mg / ml, about 0.3 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, or about 1 mg / ml). In some embodiments of the pharmaceutical composition in liquid formulation, the surfactant (such as polysorbate 80) is present in a concentration of 0.05-0.4 mg / ml. In some embodiments of the pharmaceutical composition in liquid formulation, the surfactant (such as polysorbate 80) is present in a concentration of about 0.2 mg / ml.

[0114] In some embodiments of the pharmaceutical composition in liquid formulation form, the pharmaceutical composition comprises:

[0115] The antibody-drug conjugate shown in Formula I (e.g., any ADC molecule shown in Table 1) has a concentration of 1-30 mg / ml, 5-25 mg / ml, 10-20 mg / ml, or about 15 mg / ml;

[0116] Buffers (such as histidine-histidine hydrochloride buffer) at concentrations of 1-10 mg / ml, 2-6 mg / ml, 2-4 mg / ml, or approximately 4.04 mg / ml;

[0117] Stabilizers (such as trehalose and / or sucrose) are present in concentrations of 30-110 mg / ml, 40-90 mg / ml, 40-80 mg / ml, or approximately 60 mg / ml.

[0118] Surfactants (such as polysorbate 80) are present in concentrations of 0.005-1 mg / ml, 0.05-0.4 mg / ml, or approximately 0.2 mg / ml.

[0119] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, wherein the histidine content is 0.1-1 mg / ml or 0.1-0.7 mg / ml or 0.1-0.5 mg / ml or 0.2-0.5 mg / ml or about 0.44 mg / ml, and the histidine hydrochloride content is 1-10 mg / ml or 2-6 mg / ml or 2-4 mg / ml or about 3.6 mg / ml.

[0120] In some embodiments of the pharmaceutical composition in liquid formulation, the pharmaceutical composition comprises about 15 mg / ml of an antibody-drug conjugate of Formula I (e.g., any of the ADC molecules shown in Table 1); about 4 mg / ml of a histidine-histidine hydrochloride buffer, wherein the histidine content is about 0.44 mg / ml and the histidine hydrochloride content is about 3.6 mg / ml; a stabilizer (such as trehalose and / or sucrose) at a content of about 60 mg / ml; and a surfactant (such as polysorbate 80) at a content of about 0.2 mg / ml.

[0121] In some embodiments of the pharmaceutical composition in liquid formulation, the pharmaceutical composition comprises about 15 mg / ml of an antibody-drug conjugate of Formula I (e.g., any of the ADC molecules shown in Table 1), about 0.44 mg / ml of histidine, about 3.6 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, about 60 g / L of trehalose, and the pH of the pharmaceutical composition in liquid formulation is about 5.3.

[0122] In some embodiments, the pharmaceutical composition comprises:

[0123] 1-30 or 5-25 or 10-20 or about 15 parts by weight of the antibody-drug conjugate of Formula I (e.g., any of the ADC molecules shown in Table 1);

[0124] 1-10 or 2-6 or about 4.04 parts by weight of buffer;

[0125] 30-110 or 40-90 or 40-80 or about 60 parts by weight of stabilizer (such as trehalose and / or sucrose); and

[0126] 0.005-1 or 0.05-0.4 or about 0.2 parts by weight of surfactant (such as polysorbate 80).

[0127] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, wherein histidine is 0.1-1 or 0.1-0.7 or 0.2-0.5 or about 0.44 parts by weight, and histidine hydrochloride is 1-10 or 2-6 or 2-4 or about 3.6 parts by weight.

[0128] In some embodiments, the pharmaceutical composition is a lyophilized injection, which, when diluted with water for injection, contains about 15 mg / ml of an antibody-drug conjugate of Formula I (e.g., any ADC molecule shown in Table 1), about 0.44 mg / ml of histidine, about 3.6 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, and about 60 g / L of trehalose, wherein the pH of the injection is about 5.3.

[0129] In some embodiments, the pharmaceutical composition is a lyophilized injection, the stock solution of which comprises about 15 mg / ml of an antibody-drug conjugate of Formula I (e.g., any ADC molecule shown in Table 1), about 0.44 mg / ml of histidine, about 3.6 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, and about 60 g / L of trehalose, the pH of the stock solution being about 5.3.

[0130] In some embodiments, the pharmaceutical composition is an injection (including direct injection and lyophilized reconstituted injection), the injection comprising about 15 mg / ml of an antibody-drug conjugate of Formula I (e.g., any ADC molecule shown in Table 1), about 0.44 mg / ml of histidine, about 3.6 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, and about 60 g / L of trehalose, the pH of the injection being about 5.3.

[0131] In a second aspect, this application provides a method for preparing the pharmaceutical composition described in the first aspect, the method comprising the following steps:

[0132] (1) Prepare the antibody-drug conjugate solution shown in Formula I;

[0133] (2) Preparation of ultrafiltration replacement buffer; and

[0134] (3) Change the antibody-drug conjugate prepared in step (1) to the buffer solution prepared in step (2).

[0135] In some implementations, the method further includes (4) freeze drying.

[0136] In some implementations, step (2) includes weighing the buffer and / or stabilizer according to the prescription amount, diluting it with water for injection to the target preparation volume, stirring and mixing thoroughly to obtain a displacement buffer.

[0137] In some implementations, step (3) includes using an ultrafiltration membrane to ultrafilter and replace the antibody-drug conjugate obtained in step (1) into the buffer solution obtained in step (2) by a certain volume, diluting the antibody-drug conjugate to the target concentration, and adding a surfactant by volume fraction to obtain the stock solution.

[0138] In some embodiments, the method further includes a step of sterile filtration of the resulting buffer solution.

[0139] In some implementations, step (4) freeze drying includes: (1) pre-freezing, (2) primary drying, and (3) desorption drying.

[0140] In some implementations, step (4) freeze-drying is performed under the following conditions:

[0141] (1) The pre-freezing conditions are: -60 to -30℃ or -50 to -30℃ or about -40℃, for 18 to 1800 minutes or 30 to 1500 minutes or 80 to 1000 minutes or 100 to 800 minutes or about 180 minutes;

[0142] (2) The conditions for one drying are: -25 to 0℃ or -20 to 0℃ or about -5℃, pressure 0.02-0.5mbar (absolute pressure) or about 0.15mbar, time 300 minutes to 10000 minutes or 300 minutes to 2000 minutes or about 600 minutes;

[0143] (3) The conditions for drying are: 15-45℃ or about 35℃ or about 30℃, pressure 0.1-0.5mbar (absolute pressure) or 0.25mbar, time 90-5000 minutes or 500-2000 minutes or about 900 minutes.

[0144] In some embodiments, the pharmaceutical composition of the first aspect can be used to treat proliferative diseases. In some embodiments, the proliferative disease is a cancer associated with abnormal FRα expression, including lung cancer (e.g., lung adenocarcinoma, small cell lung cancer, or non-small cell lung cancer), oral cancer, breast cancer, ovarian cancer, colon cancer, cervical cancer, uterine cancer, or endometrial cancer, etc.

[0145] Secondly, this application provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for treating proliferative diseases. In some embodiments, the proliferative disease is a cancer associated with abnormal FRα expression, including lung cancer (e.g., lung adenocarcinoma, small cell lung cancer, or non-small cell lung cancer), oral cancer, breast cancer, ovarian cancer, colon cancer, cervical cancer, uterine cancer, or endometrial cancer, etc.

[0146] Thirdly, this application provides a method for treating or preventing proliferative diseases in an individual, the method comprising administering a therapeutically effective dose of the pharmaceutical composition described in the first aspect to a patient in need. In some embodiments, the proliferative disease is a cancer associated with abnormal FRα expression, including lung cancer (e.g., lung adenocarcinoma, small cell lung cancer, or non-small cell lung cancer), oral cancer, breast cancer, ovarian cancer, colon cancer, cervical cancer, uterine cancer, or endometrial cancer, etc.

[0147] In some implementations, cancers associated with abnormal FRα expression are cancers in which FRα (FRα+) is highly expressed on the surface of cancer cells.

[0148] In some implementations, the degree of antigen expression in cancer can be characterized by the antigen positivity rate of cancer cells. For example, cancer with high FRα expression (FRα+) can be defined as at least 60% of cancer cells in a cancer cell population expressing FRα, or at least 70% of cancer cells expressing FRα, or at least 80% of cancer cells expressing FRα, or at least 90% of cancer cells expressing FRα, or at least 95% of cancer cells expressing FRα, or at least 98% of cancer cells expressing FRα, or at least 99% of cancer cells expressing FRα.

[0149] In other embodiments, the degree of FRα positivity of the tumor can be analyzed qualitatively, quantitatively, or semi-quantitatively using various methods in the art.

[0150] The pharmaceutical composition of this application can be used alone or in combination with other antitumor agents.

[0151] As demonstrated in the examples below, the exemplary pharmaceutical compositions of this application, under the example formulation conditions, are stably stored, ensuring the stability of the physicochemical properties of the antibody portion while minimizing the shedding of free drug. Stability studies showed that the physicochemical properties of the antibody portion did not change significantly, meeting the storage requirements for antibody preparations. The DAR value and DAR8 distribution of the ADC portion did not change significantly, indicating that the conjugate drug exhibits uniform quality during storage and can be stored for extended periods.

[0152] It should be understood that the specific embodiments described above and the examples described below are for the purpose of better illustrating the content of this application, but are not limited to the specific embodiments and examples described herein. This application includes various aspects, embodiments, and combinations of said aspects and / or embodiments described herein. The above description and the following examples are intended to illustrate, not limit, the scope of this application. Other aspects, improvements, and modifications within the scope of this application will be apparent to those skilled in the art. Therefore, those skilled in the art should recognize that the scope of this application also includes the improvements and modifications to the said aspects and embodiments. Example

[0153] The inventions of this application are further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this application. The preferred embodiments and materials shown herein are for illustrative purposes only.

[0154] The structures of the compounds in this application were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or high-performance liquid chromatography (HPLC). The NMR determination was performed using a Bruker Avance III 400MHz NMR spectrometer; the LC-MS was performed using a SHIMADZU LC-20AD-PDA-LCMS-2020 instrument; and the HPLC was performed using a SHIMADZU LC-20AD-PDA high-performance liquid chromatograph.

[0155] The starting materials used in the embodiments of this application are known and commercially available, or can be synthesized using methods known in the art.

[0156] The antibodies in this application can be prepared using hybridoma techniques first described by Kohler et al., Nature (1975), or using recombinant DNA methods (US Patent 4,816,567, etc.).

[0157] Preparation Examples

[0158] Explanation of Abbreviations

[0159] Preparation Example 1: Synthesis of Compound 18

[0160] Compound 11 (47.5 g, 189 mmol) and methanol (250 mL) were added to a reaction flask. 80% hydrazine hydrate (35.4 g, 567 mmol) was slowly added at room temperature. The mixture was heated to 70 °C and refluxed for 6 hours. After cooling, white crystals precipitated. The crystals were filtered, and the remaining solid was washed with methanol (20 mL × 3) to give compound 12, a white solid (47.47 g, 100% yield). MS (ESI): (M + H) + Calculated value: 252.1, Experimental value: 252.2.

[0161] Compound 12 (47.4 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol), and ethanol (400 mL) were added to a reaction flask and dissolved by stirring at room temperature. Carbon disulfide (17 g, 283 mmol) was slowly added, and the mixture was heated to 100 °C and refluxed for 5 hours. After removing the solvent under reduced pressure, water (50 mL) was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The mixture was extracted with dichloromethane (100 mL × 3), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 13 (49.9 g, 90% yield). MS (ESI): (M+H) + The calculated value is 294.1, and the experimental value is 294.3.

[0162] Compound 13 (5.86 g, 20 mmol), triethylamine (2.42 g, 24 mmol), and tetrahydrofuran (36 mL) were added to a reaction flask. Iodomethane (3.12 g, 22 mmol) was then added to the reaction mixture. The reaction was stirred at 25 °C for 1.5 hours until the reaction was complete. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to give compound 14 (4.05 g, 66% yield). MS (ESI): (M+H) + Calculated value: 308.1, experimental value: 308.2.

[0163] Compound 14 (3.3 g, 10.6 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and ethyl hydrochloride solution (2.7 mL, 4 M) was added dropwise at 25 °C. The reaction was continued for 6 hours. After removing the solvent under reduced pressure, the crude product of compound 15 was obtained and directly added to the next reaction.

[0164] The crude product of compound 15, diethylene glycol anhydride of compound 6 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask and reacted at 25 °C for 1.5 h. The solvent was removed under reduced pressure, and the residue was added to diethyl ether. After filtration, the residue was washed with water and diethyl ether to give compound 17 (3.4 g, 99% yield). MS (ESI): (M+H) + Calculated value: 324.1, experimental value: 324.2.

[0165] Compound 17 (3.4 g, 10.5 mmol) and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0 °C, and the reaction was brought back to 25 °C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. The mixture was extracted with ethyl acetate (100 mL × 3), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 18 (2.4 g, 65% yield). MS (ESI): (M + H) + Calculated value: 356.0, Experimental value: 356.0.

[0166] Preparation Example 2: Synthesis of Compound LD1 and Resolution of LD1-1 and LD1-2

[0167] At 0°C, 1-(4-aminophenyl)-3-butyn-1-ol (4997 mg, 31 mmol), N-fluorenylmethoxycarbonyl-L-alanine (9651 mg, 31 mmol), EEDQ (11498 mg, 46.5 mmol), and ultradry DCM (90 mL) were added to a reaction flask. After reacting for 3 h, the solvent was removed under reduced pressure, and 500 mL of MTBE was added to the residue for slurry mixing to give compound 72 (9007 mg, yield 64%, dr = 1:1). MS (ESI): (M+H) + Calculated value: 455.2, Experimental value: 455.2.

[0168] At 0°C, compound 72 (4000 mg, 8.8 mmol, dr = 1:1) and 100 mL of commercially available THF were added to a reaction flask. DBU (1337 mg, 8.8 mmol) was slowly added with stirring, and the reaction was allowed to proceed for half an hour before being moved to room temperature. After the starting material disappeared under TLC monitoring, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to give compound 73 (1776 mg, 87% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value: 233.1, Experimental value: 233.2.

[0169] Compound 73 (1624 mg, 7 mmol, dr = 1:1), Fmoc-Val-OSu (362 mg, 8.4 mmol, CASRN: 688585-20-8), and N,N-dimethylformamide (150 mL) were added to a reaction flask. The mixture was stirred, and DIEA (1158 μL, 7 mmol) was slowly added dropwise. The reaction was carried out at 25 °C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added and stirred. A white solid precipitated. This process was repeated three times to obtain compound 74 (dr = 1:1). MS (ESI): (M+H) + Calculated value: 554.3, experimental value: 554.4.

[0170] Compound 74 (1303 mg, 2.4 mmol, dr = 1:1), p-nitrobenzene chloroformate (964.8 mg, 4.8 mmol), and THF (120 mL) were added to a reaction flask. The mixture was stirred, and Py (382.8 μL, 4.8 mmol) was added dropwise. The reaction was carried out at 65 °C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to give compound 75 (1430 mg, 83% yield, dr = 1:1). MS (ESI): (M + Na) + Calculated value: 741.3, experimental value: 741.5.

[0171] Compound 75 (1554 mg, 2.2 mmol, dr = 1:1), compound 76 eczetcon mesylate (520 mg, 1.8 mmol), and N,N-dimethylformamide (80 mL) were added to a reaction flask. The mixture was stirred, and DIEA (740 μL, 4.5 mmol) was slowly added dropwise. The reaction was carried out at 25 °C for 20 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to give compound 77 (1442 mg, 79% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value: 1015.4, experimental value: 1015.1.

[0172] At 0°C, compound 77 (1400 mg, 1.37 mmol, dr = 1:1) and 35 mL of commercially available THF were added to a reaction flask. DBU (209 mg, 1.37 mmol) was slowly added with stirring. After reacting for half an hour, the mixture was moved to room temperature and reacted for another 40 minutes until the starting material disappeared by TLC. The reaction solution was concentrated under vacuum to remove THF, and DCM / PE (20 mL / 200 mL) was added to the residue and stirred. The mixture was filtered through a vacuum funnel to obtain compound 78 (1032 mg, 95% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value: 793.3, experimental value: 793.6.

[0173] Compound 78 (793 mg, 1 mmol, dr = 1:1), compound 18 (426 mg, 1.2 mmol), EDCI (382 mg, 2 mmol), HOBT (202 mg, 1.5 mmol), and N,N-dimethylformamide (10 mL) were added to a reaction flask and reacted at 25 °C for 1 h. The solvent was removed under reduced pressure, and the residue was purified by C18 chromatography (60% ACN / 0.05% formic acid in H2O). Lyophilization gave compound 79 (960 mg, 85% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value: 1130.4; Experimental value: 1130.7.

[0174] Compound 79 (903 mg, 0.8 mmol), compound 80 (702 mg, 1.2 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (34.7 mg, 0.08 mmol), and cuprous bromide (11.4 mg, 0.08 mmol) were added to a reaction flask, and the mixture was purged with nitrogen three times. THF / DMF / H₂O (3.5 mL:0.6 mL:0.4 mL) was added, and the reaction was carried out at 25 °C for 0.5 h. The residue was purified by C18 preparative chromatography (62% ACN / 0.05% formic acid in H₂O) to give compound LD1 (1180 mg, yield 86%, dr = 1:1). MS (ESI): (M+H)+ Calculated value: 1715.7, experimental value: 1716.2.

[0175] LD1 (1180 mg, dr = 1:1) was prepared and separated on a preparative column (Yuxu Xtimate C18 30×250 mm×10 μm) using ACN / H2O (0.05% formic acid) to obtain LD1-1 (550 mg, HPLC retention time 18.397 min) and LD1-2 (520 mg, HPLC retention time 18.163 min).

[0176] Note: HPLC instrument information: Thermo liquid chromatograph (ADC-U3000-01); Column: Hypersil GOLD TM (4.6*250mm, 5μm); Column temperature: 30℃; Injection plate temperature: 10℃; Mobile phase: A: Water + 0.05% TFA; B: ACN; Flow rate: 1.0mL / min; Detection wavelength: 254nm; Injection volume: 10μL;

[0177] Gradient condition:

[0178] Preparation Example 3: Synthesis of LD1-1 from chiral source 82 obtained by resolving racemic source 72

[0179] Chiral resolution of intermediate 72: Compound 72 (50 g) was resolved by supercritical fluid chromatography (SFC) to obtain compound 81 (19 g, retention time 13.55 min) and compound 82 (20 g, retention time 16.29 min). SFC resolution method: Column type: DAICL CHIRALCEL OD (250 mm - 50 mm, 10 μm); Mobile phase: A: CO2, B: CO2-ACN / i-PrOH (0.1% NH3H2O); Isocratic elution: B in A for 50%; Flow rate: 200 mL / min; Detector: PDA; Column temperature: 25 °C; Back pressure: 100 Bar;

[0180] HPLC Method: Instrument Information: Thermo Liquid Chromatograph (ADC-U3000-01); Column: (4.6*150mm, 5μm); Column temperature: 25℃; Injection plate temperature: 25℃; Mobile phase: A: 10mM NH4FA; B: ACN; Flow rate: 0.8mL / min; Detection wavelength: 254nm; Injection volume: 2μl;

[0181] Gradient condition:

[0182] X-ray confirmation revealed the structure and configuration of compound 82 as follows, and its single-crystal diffraction patterns are shown in Figures 1A and 1B:

[0183] Compound 82 (3632 mg, 8 mmol) and 90 mL of commercially available THF were added to a reaction flask at 0°C. DBU (1215 mg, 8 mmol) was slowly added with stirring, and the mixture was reacted for half an hour before being moved to room temperature. After the starting material disappeared under TLC monitoring, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to give compound 83 (1707 mg, 92% yield). MS (ESI): (M+H) + Calculated value: 233.1, Experimental value: 233.2.

[0184] Compound 83 (1624 mg, 7 mmol), Fmoc-Val-OSu (362 mg, 8.4 mmol), and N,N-dimethylformamide (150 mL) were added to a reaction flask. The mixture was stirred, and DIEA (1158 μL, 7 mmol) was slowly added dropwise. The reaction was carried out at 25 °C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added and stirred. A white solid precipitated. This process was repeated three times to obtain compound 84. MS (ESI): (M+H) + Calculated value: 554.3, experimental value: 554.4.

[0185] Compound 84 (1661 mg, 3 mmol), p-nitrobenzene chloroformate (1206 mg, 6 mmol), and THF (150 mL) were added to a reaction flask. The mixture was stirred, and Py (474 ​​μL, 6 mmol) was added dropwise. The reaction was carried out at 65 °C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to give compound 85 (1831 mg, 85% yield). MS (ESI): (M+H) + Calculated value: 719.8, experimental value: 719.9.

[0186] Compound 85 (1436 mg, 2 mmol), compound 86 eczetcon mesylate (1168.2 mg, 2.2 mmol), and N,N-dimethylformamide (100 mL) were added to a reaction flask. The mixture was stirred, and DIEA (695 μL, 4 mmol) was slowly added dropwise. The reaction was carried out at 25 °C for 20 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to give compound 87 (1664 mg, 82% yield). MS (ESI): (M+H) + Calculated value: 1015.4, experimental value: 1015.1.

[0187] At 0°C, compound 87 (1522 mg, 1.5 mmol) and 40 mL of commercially available THF were added to a reaction flask. DBU (228 mg, 1.5 mmol) was slowly added with stirring. After reacting for half an hour, the mixture was moved to room temperature and reacted for another 40 minutes until the starting material disappeared by TLC. The reaction solution was concentrated under vacuum to remove THF, and 15 mL of DCM and 300 mL of PE were added, producing a large amount of yellow-green solid. The solid compound 88 (1177 mg, 99% yield) was filtered out using a Buchner funnel. MS (ESI): (M+H) + Calculated value: 793.3, experimental value: 793.6.

[0188] Compounds 88 (1031 mg, 1.3 mmol), 18 (554 mg, 1.56 mmol), EDCI (498 mg, 2.6 mmol), HOBT (263 mg, 1.95 mmol), and N,N-dimethylformamide (20 mL) were added to a reaction flask and reacted at 25 °C for 1 h. The solvent was removed under reduced pressure, and the residue was purified by C18 chromatography (60% ACN / 0.05% formic acid in H2O). The residue was lyophilized to give compound 89 (1322 mg, 90% yield). MS (ESI): (M+H) + Calculated value: 1130.4; Experimental value: 1130.7.

[0189] Compound 89 (903 mg, 0.8 mmol), compound 80 (702 mg, 1.2 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (34.7 mg, 0.08 mmol), and cuprous bromide (11.4 mg, 0.08 mmol) were added to a reaction flask, and the mixture was purged with nitrogen three times. THF / DMF / H₂O (3.5 mL:0.6 mL:0.4 mL) was added, and the reaction was carried out at 25 °C for 0.5 h. The residue was purified by C18 preparative chromatography (62% ACN / 0.05% formic acid in H₂O) to give compound LD1-1 (1276 mg, 93% yield). MS (ESI): (M+H) + Calculated value: 1715.7, experimental value: 1716.2. 1H NMR (600MHz, DMSO) δ10.71(s,1H),10.08(s,1H),8.48(d,J=6.7Hz,1H),8.17(d,J=8.6Hz,3H),8.09(d,J=8.8Hz,1H),8.03(d,J=8.7Hz,2 H),7.89(s,1H),7.81(d,J=10.8Hz,1H),7.66(d,J=8.4Hz,2H),7.42–7.36(m,3H),6.61(s,1H),6.02(t,J=6.8Hz,1H),5.55(s,2H),5.38 -5.26(m,3H),4.55–4.47(m,3H),4.46–4.41(m,1H),4.36(q,J=15.6Hz,2H),4.28( s,2H),3.80(s,5H),3.62–3.55(m,36H),3.52–3.47(m,9H),3.44–3.36(m,2H),3.30 -2.29(m,1H),3.19–3.09(m,1H),2.42(s,3H),2.28–2.19(m,1H),2.17(s,3H),2.17–2.10(m ,1H),2.04–1.89(m,2H),1.42(d,J=7.0Hz,3H),1.02(d,J=6.7Hz,3H),0.98(t,J=7.2Hz,6H).

[0190] Comparison with the HPLC method of LD1-1 in Preparation Example 2 showed that the retention time of the product LD1-1 prepared in this example was consistent with that of LD1-1 in Example 2, confirming that they are the same molecule.

[0191] When compound 82 is replaced with compound 81, LD1-2 can be obtained by the same synthesis process described above.

[0192] Preparation Example 4: Synthesis of LD2-1

[0193] The difference between the synthesis processes of LD2-1 and LD1-1 lies in the final step. As shown in the figure above, in the final step of the LD2-1 synthesis, compound 89 (113 mg, 0.1 mmol), compound 61 (134 mg, 0.12 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (4.3 mg, 0.01 mmol), and cuprous bromide (14 mg, 0.01 mmol) are added to a reaction flask, the atmosphere is changed to nitrogen three times, and THF / DMF / H2O (1 mL:0.2 mL:0.2 mL) is added. The reaction is carried out at 25 °C for 0.5 hours. The residue is purified by C18 preparative chromatography (60% ACN / 0.05% formic acid in H2O) to obtain compound LD2-1 (199.7 mg, yield 89%). MS (ESI): (1 / 2 M + H) + Calculated value: 1123.0, Experimental value: 1123.0.

[0194] Similar to the differences in the synthesis processes of LD1-2 and LD1-1, when compound 82 is replaced with compound 81, LD2-2 can be obtained by following the same synthesis process, and its structure is shown below.

[0195] Preparation Example 5: Synthesis of LD3-1

[0196] The difference between the synthesis processes of LD3-1 and LD1-1 lies in the final step. As shown in the figure above, in the final step of the LD3-1 synthesis, compound 89 (113 mg, 0.1 mmol), compound 55 (147 mg, 0.12 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (4.3 mg, 0.01 mmol), and cuprous bromide (14 mg, 0.01 mmol) are added to a reaction flask, the atmosphere is changed three times with nitrogen, and THF / DMF / H2O (1 mL:0.2 mL:0.2 mL) is added. The reaction is carried out at 25 °C for 0.5 hours. The residue is purified by C18 preparative chromatography (55% ACN / 0.05% formic acid in H2O) to obtain compound LD3-1 (229 mg, yield 98%). MS (ESI): (1 / 2 M + H) + Calculated value: 1170.0, Experimental value: 1170.0.

[0197] Similar to the differences in the synthesis processes of LD1-2 and LD1-1, when compound 82 is replaced with compound 81, LD3-2 can be obtained through the same synthesis process described above, and its structure is shown below.

[0198] Preparation Example 6: ADC Preparation and DAR Measurement

[0199] Antibodies with known sequences can be obtained by using protein expression methods well known to those skilled in the art. After fermentation broth, samples with high purity can be obtained through steps such as affinity chromatography and ion chromatography.

[0200] Dilute the antibody sample to approximately 10 mg / mL with a suitable buffer (consistent with the sample buffer). Add an appropriate amount of reducing agent TCEP, adjusting the equivalent number according to the target DAR; for a target DAR of 8, add 8-10 equivalents. Then adjust the pH to 7-7.4 with Tris buffer and reduce at room temperature for 1-1.5 hours. The antibody reduction intermediate can be monitored using CE-SDS. After sufficient antibody reduction, first adjust the pH to approximately 6.5 with an appropriate amount of saturated citric acid solution, then add an excess of linker-drug (LD) in DMSO solution, making the linker-drug equivalent 15-20 times the antibody equivalent. Initiate the coupling reaction at room temperature for approximately 30 minutes. After coupling, filter the reaction solution, then use a centrifuge to concentrate and ultrafilter to remove excess linker-drug and other small molecule impurities. After purification, perform DAR determination using reversed-phase chromatography. Preparation methods for F-ADC and FS-ADC are also provided.

[0201] The concentration of farletuzumab (“F”) or farletuzumab-FcS (“FS”) antibodies was adjusted to 10 g / L using phosphate buffer (pH 7.0). After buffer replacement, the antibody solution was placed in a centrifuge tube, and 7.0 equivalents of 5 mM TCEP (Adamas-beta, Ltd.) were added. The reaction was carried out at 37°C for 1 hour to reduce the interchain disulfide bonds of the antibodies to thiol groups. Subsequently, 20 mM linker-drug dissolved in DMSO (10 equivalents of toxin per antibody) was added. The reaction was carried out at 10°C for 1 hour. After coupling, residual linker toxins were removed by ultrafiltration and the solution was stored in PBS solution at pH 7.4. The average drug loading of each antibody was determined by reverse chromatography.

[0202] By adjusting the amounts of TCEP and linker-drug, ADCs with different DAR values ​​can be obtained.

[0203] The statistical data of all ADC samples are shown in the table below.

[0204] Table 2

[0205] Preparation Example 7: Preparation and DAR Measurement of Control ADC (F-BATADC)

[0206] CB07-Exatecan was synthesized according to the method in reference CN116333135A. Following the aforementioned ADC preparation method, F-BATADC was synthesized using CB07-Exatecan as the linker drug and Farletuzumab as the antibody. The DAR value was determined to be 7.9 by reverse phase chromatography.

[0207] Test Implementation Examples

[0208] Test Example 1: Determination of ADC hydrophilicity by hydrophobic interaction chromatography (HIC)

[0209] HPLC conditions: ① Column type: TSKgel Butyl-NPR, 2.5μm, 4.6*100mm, PN:0042168; ② Column temperature: 30℃; ③ UV: 280nm; ④ Mobile phase: A: 1.5M (NH4)2SO4, 25mM NaPi, pH 6.73; B: 12.5mM NaPi (pH 7.28): 20% IPA = 80:20 (v:v); ⑤ Flow rate: 0.7mL / min; ⑥ Elution gradient: 0min→1.01min (0% B→20% B), 1.01min→10min (20% B→100% B), 10min→11min (100% B→100% B), 11min→11.01min (100% B→0% B), 11.01min→17min (0% B)

[0210] Table 3: Hydrophilicity of naked antibodies and various ADCs

[0211] Highly hydrophilic linkers can overcome the difficulties in coupling many toxins due to poor water solubility, reduce the formation of aggregates during coupling, and ensure that the ADC has a lower clearance rate and better PK. The data in Table 3 show that different hydrophilic side chains can improve the hydrophilicity of the ADC to varying degrees. Among them, the naked anti-farletuzumab has the best hydrophilicity. The hydrophilicity of each ADC in this application is superior to the control F-BATADC.

[0212] Test Example 2: Determination of antigen-binding activity of ADC by enzyme-linked immunosorbent assay (ELISA)

[0213] 1. Antigen coating: Dilute the FORL1 antigen (manufacturer: Sinocare, catalog number: 11241-H08H) to 0.1 μg / ml with coating buffer (0.05 mol / L carbonate buffer, pH 9.6), add 100 μl to each well of an ELISA-specific polystyrene microplate (manufacturer: Corning, catalog number: 3590), and incubate overnight at 4°C.

[0214] 2. Blocking: Discard the coating solution in the microplate and wash three times with washing buffer PBST (PBS containing 0.05% Tween-20 10mM, pH 7.4). Add 300 μl of 2% BSA to each well and block at 37°C for 1 h.

[0215] 3. Washing: Wash the microplate three times with washing solution, perform two replicates for each sample, and add 100 μL of Farletuzumab-FS or Farletuzumab or F-ADC1-1-8 at a maximum concentration of 1000 nM. The ADC sample was serially diluted 11 times with 2% BSA and incubated at 37°C for 2 h.

[0216] 4. Secondary antibody: Discard the FRα primary antibody diluent in the microplate, wash 3 times with washing buffer, add secondary antibody (goat anti-human IgG (L&H) secondary antibody, HRP) to each well and incubate at 37°C for 1 hour;

[0217] 5. Color development: Discard the secondary antibody dilution solution, wash 6 times with washing solution, add 100 μl of TMB color development solution, and develop the color for about 6-8 min. Then add 100 μl of 2M dilute hydrochloric acid to terminate the reaction.

[0218] 6. Plate reading: After the reaction is terminated, read the absorbance at OD450 on an ELISA reader (manufacturer: BioTek, model: Synergy LX);

[0219] 7. Calculate EC 50

[0220] Table 4: Antigen binding activity of naked antibodies and ADCs

[0221] As shown in Table 4, the antigen-binding activity of F-ADC1-1-8 of this application is comparable to that of naked anti-Farletuzumab-FS or Farletuzumab.

[0222] Test Example 3: Pharmacodynamic evaluation of each ADC in a mouse model of human oral cancer KB xenograft tumor

[0223] Using the FRα-positive cell line KB as the test cell line, 10 7 Six cells were inoculated into the right forelimb axilla of null / null mice (n=6 per group). The cells were inoculated when the average tumor volume of the mice reached 100 mm². 3 (Groups 1-6) and 145mm 3Approximately 7-8 groups were randomly assigned to receive the drug via a single intravenous injection. The day of group assignment was designated as day 0, and drug administration began on day 0. Post-tumor inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities in the eyes, fur, or other areas. Tumor volume was calculated using the formula: Tumor volume (mm²). 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0224] Table 5: Drug efficacy in groups 1-6 of the KB cell xenograft tumor model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1 - (Ti – T0) / (Ci - C0)] × 100%, where T0 and C0 are the mean tumor volumes on the day of grouping (Day 0) for the drug administration group and the solvent control group, respectively, and Ti and Ci are the mean tumor volumes on Day 30 for the drug administration group and the solvent control group, respectively; c. Compared with the tumor volume of the solvent control group.

[0225] Table 6: Efficacy of drugs in groups 7-8 in KB cell xenograft tumor model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1 - (Ti – T0) / (Ci - C0)] × 100%, where T0 and C0 are the mean tumor volumes on the day of grouping (Day 0) for the drug administration group and the solvent control group, respectively, and Ti and Ci are the mean tumor volumes on Day 20 for the drug administration group and the solvent control group, respectively; c. Compared with the tumor volume of the solvent control group.

[0226] As shown in Table 5, in the small-volume tumor model test, the tumor inhibition rate (TGI) of the ADCs in each experimental group of this application at a dose of 3 mg / kg were 104.7%, 104.7%, 104.6%, and 104.6%, respectively, which were significantly better than the control group F-BATADC (TGI 92.0%). Complete remission (CR) was achieved in 4, 4, 3, and 3 mice in experimental groups 2, 4, 5, and 6, respectively. Furthermore, there was no significant difference in efficacy between ADCs with different chiral linker configurations.

[0227] As shown in Table 6, in the large-volume tumor model test, when the dose of F-ADC1-1-8 was reduced to 1.5 mg / kg, the tumor inhibition rate (TGI) still reached 105.6%.

[0228] No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity was observed. Mice tolerated the ADC of this application well during the treatment period.

[0229] Test Example 4: Pharmacodynamic evaluation of each ADC in a mouse model of human lung adenocarcinoma NCI-H441 xenograft tumor

[0230] Using the FRα-positive cell line NCI-H441 as the test cell line, 10 7 Six cells were inoculated into the right forelimb axilla of null / null mice (n=6 per group). The cells were inoculated when the average tumor volume of the mice reached 100 mm². 3 Approximately 1-6 groups of mice were randomly assigned to receive the drug via a single intravenous injection. The day of group assignment was designated as day 0, and drug administration began on day 0. Post-tumor inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities observed in the eyes, fur, or other areas. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b) 2 (Where a represents the major axis and b represents the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0231] Table 7: Drug efficacy in each group in the NCI-H441 cell xenograft tumor model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1 - (Ti – T0) / (Ci - C0)] × 100%, where T0 and C0 are the mean tumor volumes on the day of grouping (Day 0) for the drug administration group and the solvent control group, respectively, and Ti and Ci are the mean tumor volumes on Day 32 for the drug administration group and the solvent control group, respectively; c. Compared with the tumor volume of the solvent control group.

[0232] As shown in Table 7, compared with the solvent control group, the ADCs in each experimental group of this application significantly inhibited tumor growth in the NCI-H441 model at a dose of 1 mg / kg, and the tumor-suppressing effect was significantly better than that of the control group F-BATADC. There were no animal deaths or significant weight loss in any treatment group, and no obvious drug toxicity was observed. Mice tolerated the ADCs of this application well during the treatment period.

[0233] Test Example 5: Pharmacodynamic evaluation of each ADC in a mouse model of human colon cancer SW620 xenograft tumor

[0234] Using the FRα-positive cell line SW620 as the test cell line, 10 7 Six cells were inoculated into the right forelimb axilla of null / null mice (n=6 per group). The cells were inoculated when the average tumor volume of the mice reached 130 mm². 3Approximately 1-2 groups of mice were randomly assigned to receive the drug via a single intravenous injection. The day of group assignment was designated as day 0, and drug administration began on day 0. Post-tumor inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities observed in the eyes, fur, or other areas. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0235] Table 8: Drug efficacy in each group in the SW620 cell xenograft tumor model Note: a. Data are expressed as mean ± standard error; b. TGI% = [1 - (Ti – T0) / (Ci - C0)] × 100%, where T0 and C0 are the mean tumor volumes on the day of grouping (Day 0) for the drug administration group and the solvent control group, respectively, and Ti and Ci are the mean tumor volumes on Day 32 for the drug administration group and the solvent control group, respectively; c. Compared with the tumor volume of the solvent control group.

[0236] As shown in Table 8, the F-ADC1-1-8 of this application significantly inhibited tumor growth in the SW620 model at a dose of 1.5 mg / kg. No animal deaths or significant weight loss were observed in the treatment group, and no obvious drug toxicity was observed. Mice tolerated the ADC of this application well during the treatment period.

[0237] Test Example 6: Pharmacodynamic evaluation of each ADC in a mouse model of human ovarian cancer OV-90 xenograft tumor

[0238] Using the FRα-positive cell line OV-90 as the test cell line, 10 7 Three cells were inoculated into the right forelimb axilla of 4-6 week old NCG- and NOD-SCID immunodeficient mice (n=3 per group). The cells were inoculated when the average tumor volume of the mice reached approximately 180 mm². 3 (Groups 1-3) and 145mm 3 (Groups 4-5) Mice were randomly assigned to receive the drug. The day of grouping was designated Day 0, and drug administration began on Day 0. Post-tumor inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities in the eyes, fur, or other areas. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). The pharmacodynamic evaluation of the test drug is shown in the table below.

[0239] Table 9: Drug efficacy in each group of the OV-90 xenograft tumor model

[0240] Note: a. Data are expressed as mean ± standard error; b. TGI% = [1 - (Ti – T0) / (Ci – C0)] × 100%, where T0 and C0 are the mean tumor volumes on Day 0 of the drug administration group and the solvent control group, respectively, and Ti and Ci are the mean tumor volumes on Day 11 of the drug administration group and the solvent control group, respectively; c. Tumor volume compared with the solvent control group; d. NCG immunodeficient mice; e. NOD SCID immunodeficient mice.

[0241] As shown in Table 9, compared with the solvent control group, the F-ADC1-1-8 of this application significantly inhibited tumor growth in the OV-90 model at doses of 1 / 3 / 5 mg / kg, with tumor growth inhibition rates (TGI) of 110.9%, 126.1%, and 120.7%, respectively. No animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity was observed. Mice tolerated the ADC of this application well during the treatment period.

[0242] Formulation Examples

[0243] The following examples use F-ADC1-1-8 of this application as an example to test and evaluate the effects of different formulation components and parameters on ADC formulations.

[0244] Formulation Example 1: Screening of pH and buffer system.

[0245] pH is a crucial factor affecting the stability of biological products. It regulates the charge distribution on the protein surface, thereby influencing intermolecular and intramolecular forces, conformation, and colloidal stability. It also affects the chemical stability of proteins, such as degradation reactions like deamidation. Appropriate buffer salts play a role in maintaining a stable pH level in a solution, and the type of buffer salt significantly impacts the physicochemical properties and stability of biological products.

[0246] 1. Changes in appearance and particle size

[0247] 20 mM histidine-histidine hydrochloride buffer (“H” group), citrate-sodium citrate buffer (“C” group), and succinate-sodium hydroxide buffer (“S” group) were prepared at pH 5.0, 5.5, 6.0, and 6.5. F-ADC-1-1-8 was ultrafiltered into these 12 buffers to dilute the final protein concentration to approximately 10 mg / ml. After filtration through a 0.22 μm microporous membrane, the thermal stability and particle size distribution of F-ADC-1-1-8 under different pH values ​​and buffer conditions were determined using DSC and DLS. The protein was also subjected to high temperature (40±2℃) and light irradiation (5±3℃, white light: 5000±500 Lux, UV: 90 μW / cm²). 2 Stability tests. Table 10 shows the appearance changes of the antibody-drug conjugates during high temperature and light exposure tests. At T0 and 7 days at high temperature, all 12 test groups were pale yellow and clear solutions. At 5 days of light exposure, some test groups showed significant appearance changes, with the sodium citrate test group (pH 6.0, 6.5) and the sodium succinate test group (pH 5.5, 6.0, 6.5) exhibiting turbidity.

[0248] T0 particle size data showed that, at the same pH, the particle size of the histidine-histidine hydrochloride test group was significantly smaller than that of the other two buffer test groups. After 7 days of high temperature, no significant changes in particle size were observed in any of the 12 test groups. After 5 days of light exposure, no significant changes in particle size were observed in the pH 5.0 and 5.5 histidine-histidine hydrochloride test groups, while the particle size of the pH 6.0 and 6.5 histidine-histidine hydrochloride test groups increased slightly. The particle size of both the citric acid-sodium citrate and succinic acid-sodium hydroxide test groups increased significantly. The pH 5.0 and 5.5 histidine-histidine hydrochloride buffers were the preferred buffer solutions.

[0249] Table 10 Appearance and Particle Size Results Note: H: Histidine - Histidine hydrochloride, C: Citric acid - Sodium citrate, S: Succinic acid - Sodium hydroxide

[0250] 2. SEC Changes

[0251] SEC-HPLC: Based on the high performance liquid chromatography method of General Chapter 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the analysis and detection were performed using a chromatographic column with a chromatographic gel suitable for separating proteins with molecular weight of 10-500kD as the packing material. The ratio of protein monomer peaks and high molecular weight substances (HMWS) peaks to low molecular weight substances (HMWS) peaks was calculated according to the area normalization method.

[0252] Table 11 shows the changes in SEC content under different pH and buffer salt conditions. After 7 days of high temperature, at the same pH, the histidine-histidine hydrochloride group had the highest SEC monomer content; under the same buffer salt conditions, the pH 5.0 and 5.5 groups had relatively high SEC monomer content. After 5 days of light exposure, at the same pH, the histidine-histidine hydrochloride group had the highest SEC monomer content; under the same buffer solution, the SEC monomer content decreased with increasing pH. pH 5.0 or 5.5 histidine-histidine hydrochloride (20 mM) was determined to be the preferred buffer solution.

[0253] The test results are shown in Table 11 and Figure 2:

[0254] Table 11 SEC-HPLC Detection Results Note: H: Histidine - Histidine hydrochloride, C: Citric acid - Sodium citrate, S: Succinic acid - Sodium hydroxide

[0255] Formulation Example 2: Screening of Stabilizer Types

[0256] Based on Formulation Example 1, in order to develop a lyophilized formulation, the effects of sucrose and trehalose as stabilizers and lyophilization protectants for antibody-drug conjugates were evaluated. A 20mM histidine-histidine hydrochloride buffer containing 6% sucrose or trehalose at pH 5.3 was prepared. F-ADC1-1-8 was ultrafiltered and transferred to both stabilizer buffers, adjusting the protein concentration to approximately 15 mg / ml. SEC data as shown in Table 2 indicate that both sugars have a protective effect.

[0257] Table 12 Screening of Stabilizer Types

[0258] Furthermore, the morphology diagrams of F-ADC1-1-8 sucrose freeze-dried powder and trehalose freeze-dried powder show that the lower part of the sucrose freeze-dried powder cake exhibits significant shrinkage and collapse, while the trehalose freeze-dried powder cake shows no significant shrinkage or collapse. Considering the appearance of the powder cake, trehalose was selected as the preferred freeze-drying protectant.

[0259] Formulation Example 3: Surfactant Screening

[0260] Based on Formulation Example 2, F-ADC1-1-8 was ultrafiltered and replaced in a pH 5.3 20mM histidine-histidine hydrochloride 6% trehalose buffer to dilute the protein concentration to approximately 15 mg / ml, obtaining the formulation solution. 0.005%, 0.02%, and 0.04% polysorbate 80 were added to this solution, with a control group without added surfactant. After filtration through a 0.22 μm microporous membrane, four groups of samples with different surfactant contents were prepared. Sub-visible particle data were measured using Flowcam after 0, 3, and 6 freeze-thaw cycles (-20℃ to room temperature).

[0261] Table 13 shows the changes in non-spherical particles (NSPs) after freeze-thaw tests with different amounts of surfactant. After 3 and 6 freeze-thaw cycles, the number of NSPs in the unsurfactant group increased significantly, while no significant increase was observed in the three groups with surfactant. Different amounts of polysorbate 80 significantly inhibited the formation of subvisible particles. Considering the stability of polysorbate 80, adding 0.02% polysorbate 80 was selected as the preferred surfactant addition.

[0262] Table 13. Data on the variation of non-spherical subvisible particles (NSP) in freeze-thaw tests. Note: The particle size in the table refers to the equivalent sphere diameter (ESD).

[0263] Through the aforementioned experiments, the optimized formulation of F-ADC1-1-8 was finally confirmed to be: protein concentration 15 mg / ml, pH 5.3, 20 mM histidine-histidine hydrochloride, 0.02% (w / v) polysorbate 80, and 6.0% (w / v) trehalose.

[0264] The stock solution was prepared using the following steps:

[0265] Weigh the buffer and stabilizer according to the aforementioned prescription, dilute with water for injection to the target preparation volume, stir and mix evenly to obtain ultrafiltration replacement buffer; use an ultrafiltration membrane to ultrafilter and replace F-ADC1-1-8 into the prepared ultrafiltration replacement buffer at a certain volume, dilute the antibody-drug conjugate with water for injection to the target concentration, and add surfactant according to mass fraction or volume fraction to obtain the stock solution.

[0266] Formulation Example 4: Protein Concentration Screening

[0267] Nine buffer solutions were prepared. F-ADC1-1-8 ultrafiltration was used to replace these nine buffer solutions to dilute the protein concentration to the desired level. After filtration through a 0.22 μm microporous membrane, the samples underwent a high-temperature (40℃±2℃) screening test to determine the SEC value. Two replicate samples were set for each test point to calculate the within-group error. Analysis of variance was performed on the monomeric SEC data from the high-temperature test. An F-test was conducted at a 95% confidence level, and the p-value for each factor was calculated to select the optimal levels for the three experimental factors. As shown in Table 14, a protein concentration of 10-20% yielded ideal SEC stability.

[0268] Table 14 shows the results of the SEC single-unit variance analysis and significance test at high temperature for 7 days.

[0269] Table 14 Results of SEC single-unit variance analysis and significance test at high temperature for 7 days

[0270] Formulation Example 5: Lyophilization Process Development

[0271] Using 15 mg / ml antibody-drug conjugate (F-ADC1-1-8), 0.44 g / L histidine, 3.6 g / L histidine hydrochloride, 0.2 g / L polysorbate 80, and 60 g / L trehalose as the base formulation, the buffer and stabilizer were weighed and diluted with water for injection to the target preparation volume. The mixture was stirred and stirred evenly to obtain the ultrafiltration replacement buffer. The prepared antibody-drug conjugate was then replaced with the prepared buffer solution.

[0272] The glass transition temperature and disintegration temperature of the sample were determined. Low-temperature DSC analysis showed that the glass transition temperature of the sample was -28.66℃, and lyophilization microscopy showed that the disintegration temperature was -24.8℃.

[0273] Pre-freezing: Based on the glass transition temperature, the preliminary pre-freezing temperature for the small-scale test was set at -40℃. Before reaching -40℃, the plate was first brought to 4℃ over 30 minutes and maintained at 4℃ for another 30 minutes to ensure that all samples could be cooled from 4℃. A rapid freezing method of 2℃ / min was then used to cool to -40℃ and held for 180 minutes to ensure that all samples were fully frozen.

[0274] Single-stage drying: After pre-freezing, the samples undergo a single-stage drying process to remove over 90% of the water from the product through sublimation. To improve the sublimation rate, the plate temperature can be set above the product's allowable disintegration temperature, provided that the temperature of the unsublimated portion does not exceed its disintegration temperature. Three batches of freeze-drying tests were conducted under different conditions and with different vacuum settings.

[0275] All freeze-dried powders obtained with different primary drying parameters were white and loose, with a moisture content below 1%. The third batch (-5℃, 0.20 mbar) showed shrinkage and indentation at the bottom, while the other two batches maintained good morphology. The second batch (-5℃, 0.15 mbar) required 600 minutes less drying time in its primary drying stage compared to the first batch. Based on comprehensive analysis, the second batch process (-5℃, 0.15 mbar) was selected as the primary drying process for freeze-drying.

[0276] Table 15 Development parameters and moisture data for the primary drying process

[0277] Desorption drying: After the first drying cycle, desorption drying was carried out at 35℃ and 0.20mbar for 900min.

[0278] Key quality parameters of the final freeze-dried sample (-5℃, 0.15mbar as a single drying process) were compared before and after freeze-drying. As shown in Table 16, the product quality did not change significantly after freeze-drying, and the freeze-drying process did not have a significant impact on the product quality.

[0279] Table 16 Comparison of mass between reconstituted and original solutions of lyophilized powder

[0280] Formulation Example 6: Bioactivity Assay

[0281] Using the reconstituted solution of the F-ADC1-1-8 lyophilized formulation obtained in Formulation Example 5, the bioactivity of the reconstituted solution was determined according to the method for determining bioactivity described in the aforementioned Test Examples section. The results showed that the bioactivity of the reconstituted solution was comparable to that of the ADC alone.

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate of Formula I: Where A is an antibody or its antigen-binding fragment targeting folate receptor α (FRα); R is selected from... in, Ra is selected from C1-C3 alkylene (e.g., methylene, ethylene, n-propylene, or isopropylene), Rb is selected from C1-C3 alkyl (e.g., methyl, ethyl, n-propyl, or isopropyl); r, t, and u are each independently selected from integers in the range of 1-50, preferably integers in the range of 4-30; m is an integer or decimal in the range of 1 to 12, preferably an integer or decimal in the range of 4-8; and The pharmaceutical composition is a liquid formulation, a lyophilized formulation, or a powder for injection, preferably a liquid formulation or a lyophilized formulation, more preferably an injectable formulation, further preferably an injectable solution or a lyophilized injection, and most preferably a lyophilized injection.

2. The pharmaceutical composition of claim 1, wherein the antibody-drug conjugate of formula I has the following structure:

3. The pharmaceutical composition of claim 2, wherein the antibody-drug conjugate of formula I has the following structure:

4. The pharmaceutical composition according to any one of claims 1-3, wherein The antibody or antigen-binding fragment of the A-representing target FRα includes a heavy chain variable region and / or a light chain variable region. The heavy chain variable region includes three complementarity-determining regions (CDRs), wherein the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:3; the light chain variable region includes three complementarity-determining regions (CDRs), wherein the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID NO:6, wherein the CDRs are determined according to the Kabat numbering rules. Preferably, the amino acid sequence of the heavy chain variable region (VH) is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region (VL) is as shown in SEQ ID NO:8; More preferably, the antibody targeting FRα comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:9 or 11, and a light chain with an amino acid sequence as shown in SEQ ID NO:

10.

5. The pharmaceutical composition according to any one of claims 1-4, wherein the antibody-drug conjugate of formula I has the following structure: Where m is an integer or decimal in the range of 2-8, preferably 2, 4, 6, or 8; and Farletuzumab is an antibody targeting FRα that includes two identical heavy and light chains. Its heavy chain amino acid sequence is shown in SEQ ID NO:9, and its light chain amino acid sequence is shown in SEQ ID NO:

10. Farletuzumab-FcS is an antibody targeting FRα that includes two identical heavy and light chains. Its heavy chain amino acid sequence is shown in SEQ ID NO:11, and its light chain amino acid sequence is shown in SEQ ID NO:

10.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the pharmaceutical composition is a lyophilized injection, optionally containing residual moisture.

7. The pharmaceutical composition according to any one of claims 1-6, wherein, based on 100 wt% of the pharmaceutical composition, the mass percentage of the antibody-drug conjugate represented by Formula I is 1-30%, preferably 5-25% or 10-25%, for example about 19%.

8. The pharmaceutical composition according to any one of claims 1-7, further comprising a buffer; preferably, the buffer is selected from one or more of acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, tromethamine (Tris), and morpholine ethanesulfonic acid (MES), more preferably a histidine buffer, and even more preferably a histidine-histidine hydrochloride buffer.

9. The pharmaceutical composition of claim 8, wherein the buffer content is 1-10% by mass, preferably 1-9.9% or 2.5-7.5%, for example about 5%, based on 100 wt% of the pharmaceutical composition.

10. The pharmaceutical composition of claim 8 or 9, wherein the buffer is a histidine-histidine hydrochloride buffer, and based on 100 wt% of the pharmaceutical composition, the mass percentage of histidine is 0.01-2%, preferably 0.25-0.75%, for example about 0.5%, and the mass percentage of histidine hydrochloride is 1-10%, preferably 2-7%, for example about 4.5%.

11. The pharmaceutical composition according to any one of claims 1-10, further comprising a stabilizer; preferably, the stabilizer is selected from one or more of sugars (e.g., sucrose and / or trehalose), polyols (e.g., mannitol and / or sorbitol), and amino acids (e.g., L-serine, monosodium glutamate, alanine, glycine, and / or sarcosine), preferably sugars, and more preferably sucrose and / or trehalose.

12. The pharmaceutical composition of claim 11, wherein the stabilizer comprises 1-90% by mass, preferably 50%-90%, for example about 75.7%, based on 100 wt% of the pharmaceutical composition.

13. The pharmaceutical composition according to any one of claims 1-12, further comprising a surfactant; preferably, the surfactant is selected from polysorbates, more preferably polysorbate 20 and / or polysorbate 80.

14. The pharmaceutical composition of claim 13, wherein the surfactant comprises 0.01-1% by mass, preferably 0.05-0.4%, for example about 0.3%, based on 100 wt% of the pharmaceutical composition.

15. The pharmaceutical composition according to any one of claims 1-14, wherein, by weight percentage, the pharmaceutical composition comprises: The antibody-drug conjugate shown in Formula I (e.g., any antibody-drug conjugate according to claim 5) has a mass percentage of 1-30%, preferably 5-25%, more preferably 10-25%, for example about 19%; The buffer has a mass percentage of 1-10%, preferably 2.5-7.5%, for example about 5% (for example, the buffer is a histidine-histidine hydrochloride buffer, preferably wherein the mass percentage of histidine is 0.1-1%, preferably 0.25-0.75%, for example about 0.5%, and the mass percentage of histidine hydrochloride is 1-10%, preferably 1-9.9%, more preferably 2-7%, for example about 4.5%); Stabilizers (e.g., trehalose and / or sucrose), at a mass percentage of 1-90%, preferably 50%-90%, for example about 75.7%; and The surfactant (e.g., polysorbate 80) is present in a mass percentage of 0.01-1%, preferably 0.05-0.4%, for example, about 0.3%. Optionally, the pharmaceutical composition contains residual moisture.

16. The pharmaceutical composition according to any one of claims 1-15, wherein, by weight percentage, the pharmaceutical composition comprises: The antibody-drug conjugate shown in Formula I (e.g., any antibody-drug conjugate according to claim 5) has a mass percentage of about 19%; The buffer has a mass percentage of about 5% (for example, the buffer is a histidine-histidine hydrochloride buffer, wherein the mass percentage of histidine is about 0.5% and the mass percentage of histidine hydrochloride is about 4.5%). Stabilizers (e.g., trehalose and / or sucrose), at a mass percentage of approximately 75.7%; and Surfactants (such as polysorbate 80) are present in a mass percentage of approximately 0.3%. Optionally, the pharmaceutical composition contains residual moisture.

17. The pharmaceutical composition of any one of claims 1-5, wherein the pharmaceutical composition is a lyophilized injection, the lyophilized injection being used by dissolving it in a solvent (preferably water, more preferably water for injection) to obtain a solution, the lyophilized injection being obtained by lyophilizing a stock solution formed by lyophilizing a solvent (preferably ethanol, methanol or water) containing a predetermined amount of the pharmaceutical component.

18. The pharmaceutical composition according to any one of claims 1-5, wherein the pharmaceutical composition is a liquid formulation, preferably an injection.

19. The pharmaceutical composition according to any one of claims 17-18, wherein the content of the antibody-drug conjugate of Formula I in the solution, stock solution and / or liquid formulation is 1-30 mg / ml, preferably 5-25 mg / ml, more preferably 10-20 mg / ml, for example about 15 mg / ml.

20. The pharmaceutical composition according to any one of claims 17-19, wherein the solution, stock solution and / or liquid formulation further comprises a buffer; preferably, the buffer is selected from one or more of acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, tromethamine (Tris), and morpholine ethanesulfonic acid (MES), more preferably a histidine buffer, and even more preferably a histidine-histidine hydrochloride buffer.

21. The pharmaceutical composition of claim 20, wherein the content of the buffer (e.g., histidine-histidine hydrochloride buffer) is 1-10 mg / ml, preferably 2-6 mg / ml, for example about 4.04 mg / ml.

22. The pharmaceutical composition of claim 20 or 21, wherein the buffer is a histidine-histidine hydrochloride buffer, the histidine content is 0.1-1 mg / ml, preferably 0.1-0.7 mg / ml or 0.2-0.5 mg / ml, for example about 0.44 mg / ml, and the histidine hydrochloride content is 1-10 mg / ml, preferably 2-6 mg / ml or 2-4 mg / ml, for example about 3.6 mg / ml.

23. The pharmaceutical composition according to any one of claims 17-22, wherein the solution, stock solution and / or liquid formulation further comprises a stabilizer; preferably, the stabilizer is selected from one or more of sugars (e.g., sucrose and / or trehalose), polyols (e.g., mannitol and / or sorbitol), and amino acids (e.g., L-serine, monosodium glutamate, alanine, glycine and / or sarcosine), more preferably sugars, and even more preferably sucrose and / or trehalose.

24. The pharmaceutical composition of claim 23, wherein the stabilizer (e.g., sucrose and / or trehalose) is present in an amount of 30-110 mg / ml, preferably 40-90 mg / ml or 40-80 mg / ml, for example about 60 mg / ml.

25. The pharmaceutical composition of any one of claims 17-24, wherein the solution, stock solution and / or liquid formulation further comprises a surfactant; preferably, the surfactant is selected from polysorbates, and more preferably polysorbate 20 and / or polysorbate 80.

26. The pharmaceutical composition of claim 25, wherein the surfactant (e.g., polysorbate 80) is present in an amount of 0.005-1 mg / ml, preferably 0.05-0.4 mg / ml, for example about 0.2 mg / ml.

27. The pharmaceutical composition according to any one of claims 17-26, wherein the solution, stock solution and / or liquid formulation has a pH of 4.0-7.5 (e.g., about 4.0, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.5, about 7.0 or about 7.5), preferably 4.5-7.0, more preferably 4.5-5.5, further preferably 5.0-5.5, and most preferably about 5.

3.

28. The pharmaceutical composition of any one of claims 17-27, wherein the solution, stock solution, and / or liquid formulation comprises: The antibody-drug conjugate shown in Formula I (e.g., any antibody-drug conjugate according to claim 5) has a content of 1-30 mg / ml, preferably 5-25 mg / ml or 10-20 mg / ml, for example about 15 mg / ml; A buffer, with a content of 1-10 mg / ml, preferably 2-6 mg / ml, for example about 4.04 mg / ml (for example, the buffer is a histidine-histidine hydrochloride buffer, preferably, the histidine content is 0.1-1 mg / ml, preferably 0.1-0.7 mg / ml or 0.2-0.5 mg / ml, for example about 0.44 mg / ml, and the histidine hydrochloride content is 1-10 mg / ml, preferably 2-6 mg / ml or about 2-4 mg / ml, for example about 3.6 mg / ml); Stabilizers (e.g., trehalose and / or sucrose) are present in a concentration of 30-110 mg / ml, preferably 40-90 mg / ml or 40-80 mg / ml, for example, about 60 mg / ml; and Surfactant (e.g., polysorbate 80) in a concentration of 0.005-1 mg / ml, preferably 0.05-0.4 mg / ml, for example about 0.2 mg / ml.

29. The pharmaceutical composition of any one of claims 17-28, wherein the solution, stock solution, and / or liquid formulation comprises about 15 mg / ml of an antibody-drug conjugate of formula I (e.g., any antibody-drug conjugate of claim 5), about 0.44 mg / ml of histidine, about 3.6 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, and about 60 g / L of trehalose, and the pH of the solution, stock solution, and / or liquid formulation is about 5.

3.

30. The pharmaceutical composition of any one of claims 17-28, wherein the solution, stock solution, and / or liquid formulation comprises: 1-30 (preferably 5-25 or 10-20, for example about 15) parts by weight of the antibody-drug conjugate of Formula I (e.g. any antibody-drug conjugate of claim 5); 1-10 (preferably 2-6, for example about 4.04) parts by weight of a buffer, preferably, the buffer is a histidine-histidine hydrochloride buffer, wherein histidine is 0.1-1 (preferably 0.1-0.7 or 0.2-0.5, for example about 0.44) parts by weight and histidine hydrochloride is 1-10 (preferably 2-6 or about 2-4, for example about 3.6) parts by weight; 30-110 (preferably 40-90 or 40-80, e.g., about 60) parts by weight of stabilizer (e.g., trehalose and / or sucrose); and 0.005-1 (preferably 0.05-0.4, for example about 0.2) parts by weight of surfactant (e.g., polysorbate 80).

31. The pharmaceutical composition according to any one of claims 1-30, for treating proliferative diseases, preferably, said proliferative disease is a disease associated with abnormal expression of FRα, such as lung cancer (e.g., lung adenocarcinoma, small cell lung cancer or non-small cell lung cancer), oral cancer, breast cancer, ovarian cancer, colon cancer, cervical cancer, uterine cancer or endometrial cancer.

32. A method for preparing a pharmaceutical composition according to any one of claims 1-30, comprising the following steps: (1) Preparation of the antibody-drug conjugate shown in Formula I; (2) Preparation of ultrafiltration replacement buffer; and (3) Change the antibody-drug conjugate prepared in step (1) to the buffer solution prepared in step (2); Preferably, step (2) includes weighing the buffer and / or stabilizer according to the prescription amount, diluting it with water for injection to the target preparation volume, stirring and mixing evenly to obtain an ultrafiltration replacement buffer; step (3) includes using an ultrafiltration membrane to ultrafilter and replace the antibody-drug conjugate obtained in step (1) into the ultrafiltration replacement buffer obtained in step (2) by a certain volume, diluting the antibody-drug conjugate with water for injection to the target concentration, and adding a surfactant according to a mass fraction or volume fraction to obtain the stock solution; optionally, the method further includes lyophilizing the stock solution, preferably, lyophilization includes: (1) Pre-freezing, (2) One-time drying, and (3) Desorption drying; Preferably, the freeze-drying step is performed under the following conditions: (1) The pre-freezing conditions are: -60 to -30°C, preferably -50 to -30°C, for example, about -40°C, for 18 to 1800 minutes, preferably 30 to 1500, 80 to 1000, or 100 to 800 minutes, for example, about 180 minutes; (2) The conditions for primary drying are: -25 to 0°C, preferably -20 to 0°C, for example about -5°C, pressure 0.02-0.5 mbar (absolute pressure), preferably 0.08-0.2 mbar, for example about 0.15 mbar, and time 200 minutes to 10000 minutes, preferably 300 to 3000 minutes or 300 to 2200 minutes, for example about 600 minutes; (3) The conditions for desorption drying are: 15-45°C, preferably about 35°C or about 30°C, pressure 0.1-0.5 mbar (absolute pressure), preferably 0.20 or 0.25 mbar, and time 90-5000 minutes, preferably 500-2000 minutes, for example about 900 minutes.

33. Use of the pharmaceutical composition according to any one of claims 1-30 in the preparation of a medicament for treating proliferative diseases, preferably, the proliferative disease being a disease associated with abnormal expression of FRα, such as lung cancer (e.g., lung adenocarcinoma, small cell lung cancer, or non-small cell lung cancer), oral cancer, breast cancer, ovarian cancer, colon cancer, cervical cancer, uterine cancer, or endometrial cancer.

34. A method for treating or preventing proliferative diseases, the method comprising administering to a patient in need of a therapeutically effective dose of the pharmaceutical composition of any one of claims 1-30; preferably, the proliferative disease is a cancer associated with abnormal expression of FRα, such as lung cancer (e.g., lung adenocarcinoma, small cell lung cancer, or non-small cell lung cancer), oral cancer, breast cancer, ovarian cancer, colon cancer, cervical cancer, uterine cancer, or endometrial cancer.

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