Pharmaceutical composition of antibody-drug conjugate

Abstract

Provided are a pharmaceutical composition of an antibody-drug conjugate, a preparation method therefor, and use thereof, wherein the antibody is an antibody or an antigen-binding fragment thereof targeting B7 homologue 3 (B7H3), and the pharmaceutical composition is a liquid formulation, a lyophilized formulation, or a powder injection formulation.

Description

A pharmaceutical composition of an antibody-drug conjugate

[0001] Citation of relevant applications

[0002] This application claims priority to Chinese Patent Application No. 202411930630.9, filed on December 26, 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, 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] B7 homologue 3 (B7H3) is a type I transmembrane protein belonging to the B7 immune co-stimulatory and co-inhibitory family. Studies have confirmed that B7H3 is widely expressed in various malignant tumors, and immunotherapeutic strategies targeting B7H3, such as antibodies, bispecific antibodies, ADCs, and CAR-T therapy, have been widely reported.

[0006] In the study of antibodies and antibody-drug conjugates (ADCs), the formation of aggregates and the generation of breakdown 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 the generation of breakdown products when formulating related formulations. Particularly when studying antibody-drug conjugate formulations, 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 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 Ab is an antibody or its antigen-binding fragment targeting B7 homology 3 (B7H3).

[0011] R is selected from C 1-6 Alkyl group, where n is an integer or decimal in the range of 1-8, and

[0012] The pharmaceutical composition is a liquid preparation, a lyophilized preparation, or a powder for injection.

[0013] Secondly, this application provides a method for preparing the pharmaceutical composition described in the first aspect, comprising the following steps:

[0014] (1) Preparation of the antibody-drug conjugate shown in Formula I;

[0015] (2) Preparation of ultrafiltration replacement buffer; and

[0016] (3) Change the antibody-drug conjugate prepared in step (1) to the buffer solution prepared in step (2).

[0017] 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.

[0018] 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.

[0019] Detailed description of the invention

[0020] Terminology Definition

[0021] 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.

[0022] 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.

[0023] 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".

[0024] This application names exemplary ADC molecules using the format of antibody code-ADC adapter-drug compound code-expected DAR. For example, "43B-JSSW-6" indicates that the ADC molecule contains "43B" as the antibody portion, contains JSSW (JSSW-001 in this example) as the adapter-drug compound, and has an expected conjugation DAR of 6. 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 results of Preparation Example 4 show that the measured / actual DAR of "43B-JSSW-6" is 5.6, which is for the ADC sample and does not contradict the expected DAR. Based on these results, those skilled in the art can also determine that the "43B-JSSW-6" sample of Preparation Example 4 mainly contains ADC molecules with a DAR of 6.

[0025] The linker-drug compound and the antibody described in this application can be linked by conventional conjugation methods in the art, including: lysine conjugation, reductive disulfide bond conjugation between the light and heavy chains, and directional conjugation (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 reductive disulfide bond conjugation between the light and heavy chains, i.e., the linking 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).

[0026] 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.

[0027] 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.

[0028] 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.).

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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 B7H3 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In a first aspect, this application provides a pharmaceutical composition comprising an antibody-drug conjugate of Formula I:

[0037] Where Ab is an antibody or its antigen-binding fragment targeting B7 homology 3 (B7H3).

[0038] R is selected from C 1-6 Alkyl group, where n is an integer or decimal in the range of 1-8, and

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

[0040] In some implementations, n is selected from an integer or decimal in the range of 4-8.

[0041] In some implementations, when n is an integer selected from the range 1-8, it can be 1, 2, 3, 4, 5, 6, 7, or 8.

[0042] In some implementations, when n is a decimal, it represents the average number of linker-drug molecules conjugated to each antibody unit in the ADC molecule contained in the pharmaceutical composition.

[0043] In some implementations, R is selected from C 1-3 alkyl.

[0044] In some embodiments, R is selected from methyl, ethyl, propyl, or isopropyl.

[0045] In some implementations, R is selected from methyl.

[0046] In some embodiments, the antibody-drug conjugate shown in Formula I has the structure shown in Formula Ia or Formula Ib:

[0047] In Equations Ia and Ib, Ab, R, and n are defined as in Equation I.

[0048] In some embodiments, the antibody-drug conjugate shown in Formula I has the structure shown in Formula I-1, Ia-1, or Ib-1:

[0049] In Equations I-1, Ia-1, and Ib-1, the definitions of Ab and n are as in Equation I.

[0050] In some embodiments, the antibody or antigen-binding fragment of B7H3 represented by Ab 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.

[0051] In some embodiments, the antibody or antigen-binding fragment of B7H3 represented by Ab 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 light chain 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. 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.

[0052] In some embodiments, the antibody Ab representing targeting B7H3 comprises a heavy chain and / or a light chain, wherein the heavy chain contains a heavy chain variable region (VH) sequence as shown in SEQ ID NO:7, and the light chain contains a light chain variable region (VL) sequence as shown in SEQ ID NO:8.

[0053] In some implementations, antibodies targeting B7H3 also include a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0054] In some implementations, the antigen-binding fragment of the antibody targeting B7H3 is selected from Fab, Fab', Fab'-SH, Fv, scFv, or F(ab')2.

[0055] In some implementations, the antibody targeting B7H3 or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0056] In some embodiments, the antibody targeting B7H3 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:10, and the amino acid sequence of which is shown in SEQ ID NO:11.

[0057] In some implementations, the antibody targeting B7H3 is antibody 43A or 43B or a biosimilar thereof, as described below.

[0058] Antibody 43A comprises two identical heavy and light chains, the amino acid sequence of the heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0059] 43A heavy chain (SEQ ID NO:9)

[0060] 43A light chain (SEQ ID NO:11)

[0061] The amino acid sequences indicated in bold are heavy / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named 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.

[0062] HCDR1: DYFMN (SEQ ID NO:1)

[0063] HCDR2:DVNPKTGSPSYNQKFKG(SEQ ID NO:2)

[0064] HCDR3:RYGFLYSMDY(SEQ ID NO:3)

[0065] LCDR1:RASQDISNYLN(SEQ ID NO:4)

[0066] LCDR2: YTSRLHS (SEQ ID NO:5)

[0067] LCDR3:QQGNTHPFT(SEQ ID NO:6)

[0068] The underlined amino acid sequences are the heavy / light chain variable region sequences, and VH and VL are named SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0069] 43AVH:

[0070] 43AVL:

[0071] 43B comprises two identical heavy and light chains, the amino acid sequence of the heavy chain is shown in SEQ ID NO:10, and the amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0072] 43B heavy chain (SEQ ID No:10)

[0073] 43B light chain (SEQ ID NO:11)

[0074] The amino acid sequences indicated in bold are heavy / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named 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.

[0075] HCDR1: DYFMN (SEQ ID NO:1)

[0076] HCDR2:DVNPKTGS PSYNQKFKG(SEQ ID NO:2)

[0077] HCDR3:RYGFLYSMDY(SEQ ID NO:3)

[0078] LCDR1:RASQDISNYLN(SEQ ID NO:4)

[0079] LCDR2: YTSRLHS (SEQ ID NO:5)

[0080] LCDR3:QQGNTHPFT(SEQ ID NO:6)

[0081] The underlined amino acid sequences are the heavy / light chain variable region sequences, and VH and VL are named SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0082] 43B VH:

[0083] 43B VL:

[0084] 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):

[0085] Table 1

[0086] Where d is an integer or decimal in the range of 2-8, preferably selected from 2, 4, 6 or 8.

[0087] 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.

[0088] Lyophilized injections are solid injections (i.e., 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 drug component in a solvent (preferably ethanol, methanol, or water).

[0089] 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.

[0090] In some embodiments, the pharmaceutical composition further comprises a buffer.

[0091] In some embodiments, the pharmaceutical composition further comprises a stabilizer.

[0092] In some embodiments, the pharmaceutical composition further comprises a surfactant.

[0093] In some embodiments, the pharmaceutical composition further comprises any two or all three of a buffer, a stabilizer, and a surfactant.

[0094] In some embodiments, the pharmaceutical composition further comprises water (for lyophilized formulations, this refers to residual moisture from the lyophilization process).

[0095] In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate of Formula I, a buffer, a stabilizer, and a surfactant.

[0096] 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 ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, or ADC-6 of this application.

[0097] 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.

[0098] 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 citrate-sodium citrate.

[0099] In some implementations, the buffer is a histidine-histidine hydrochloride buffer.

[0100] In some embodiments, the stabilizer is selected from: 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).

[0101] In some embodiments, the surfactant is selected from polysorbates (e.g., polysorbate 20 and / or polysorbate 80).

[0102] In the embodiments described below regarding pH, mass percentage (in mass %), and content (in mass / volume (e.g., g / L)), 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 may be water for injection); unless otherwise specified, when describing mass percentage, it is based on a total weight of 100 wt% of the pharmaceutical composition 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.

[0103] 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.

[0104] 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.

[0105] In some embodiments, the pharmaceutical composition is a lyophilized injection, optionally containing residual moisture.

[0106] 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% or 10-20% by mass. In some embodiments, the antibody-drug conjugate of Formula I in the pharmaceutical composition comprises about 19.38% by mass.

[0107] In some embodiments, the pharmaceutical composition contains the buffer (such as a histidine-histidine hydrochloride buffer) at a mass percentage of 1.01-12% (e.g., about 1.01%, 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%, about 10%, about 11%, or about 12%). In some embodiments, the pharmaceutical composition contains the buffer (such as a histidine-histidine hydrochloride buffer) at a mass percentage of 2-6% or 3-5%. In some embodiments, the buffer (such as histidine-histidine hydrochloride buffer) in the pharmaceutical composition comprises about 3%, about 4%, about 5%, or about 2.93% by mass.

[0108] In some embodiments, the buffer in the pharmaceutical composition is a histidine-histidine hydrochloride buffer, 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 histidine content is 0.25-0.75% or 0.3-0.5% by mass. In some embodiments, the histidine content is about 0.3%, about 0.4%, about 0.5%, or about 0.33% by mass.

[0109] 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 histidine hydrochloride is present in a mass percentage of 1.8-5.4% or 2.5-4.5%. In some embodiments, the mass percentage of histidine hydrochloride is about 2.5%, about 3.5%, about 4.5%, or about 2.60%.

[0110] In some embodiments, the stabilizer (such as trehalose and / or sucrose) in the pharmaceutical composition comprises 1-90% by weight (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 and / or sucrose) comprises 30%-90% or 35-80% by weight. In some embodiments, the stabilizer (such as trehalose and / or sucrose) comprises about 39%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 77.50% by weight.

[0111] In some embodiments, the surfactant (such as polysorbate 20 and / or polysorbate 80) 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%, about 1%). In some embodiments, the surfactant (such as polysorbate 20 and / or polysorbate 80) comprises 0.05-0.4% by mass. In some embodiments, the surfactant (such as polysorbate 20 and / or polysorbate 80) comprises about 0.1%, about 0.2%, about 0.3%, or about 0.19% by mass.

[0112] In some embodiments, the pharmaceutical composition comprises, by weight percentage:

[0113] The antibody-drug conjugate shown in Formula I (e.g., any of the ADC molecules shown in Table 1) has a mass percentage of 1-30%, 5-25%, 10-25%, 10-20%, or about 19.38%.

[0114] Buffers (such as histidine-histidine hydrochloride buffers) with a mass percentage of 1-12%, 2-6%, 3-5%, about 3%, about 4%, about 5%, or about 2.93%.

[0115] Stabilizers (e.g., trehalose and / or sucrose) at a mass percentage of 1-90%, 30%-90%, 35-80%, about 39%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 77.50%; and

[0116] Surfactants (e.g., polysorbate 20 and / or polysorbate 80) are present in a mass percentage of 0.01-1%, 0.05-0.4%, about 0.1%, about 0.2%, about 0.3%, or about 0.19%.

[0117] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, wherein the histidine mass percentage is 0.01-2% or 0.25-0.75% or 0.2-0.6% or 0.3-0.5% or about 0.3% or about 0.4% or about 0.5% or about 0.33%, and the histidine hydrochloride mass percentage is 1-10% or 1.8-5.4% or 2.5-4.5% or about 2.5% or about 3.5% or about 4.5% or about 2.60%.

[0118] In some embodiments, the pharmaceutical composition comprises, by weight percentage:

[0119] 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 19.38%.

[0120] The buffer has a mass percentage of approximately 2.93%.

[0121] Stabilizers (e.g., trehalose and / or sucrose), at a mass percentage of approximately 77.50%; and

[0122] Surfactants (e.g., polysorbate 20 and / or polysorbate 80) are present in a mass percentage of approximately 0.19%.

[0123] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, wherein the mass percentage of histidine is about 0.33% and the mass percentage of histidine hydrochloride is about 2.60%.

[0124] 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 g / L (e.g., about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 8.5 g / L, about 9 g / L, about 9.5 g / L, about 1 g / L, about 1 g / L). The concentrations of the antibody-drug conjugate shown in Formula I are approximately 0 g / L, about 10.5 g / L, about 11 g / L, about 12 g / L, about 13 g / L, about 14 g / L, about 15 g / L, about 16 g / L, about 17 g / L, about 18 g / L, about 19 g / L, about 20 g / L, about 21 g / L, about 22 g / L, about 23 g / L, about 24 g / L, about 25 g / L, about 26 g / L, about 27 g / L, about 28 g / L, about 29 g / L, or about 30 g / L. In some embodiments of the pharmaceutical composition in liquid formulation, the concentration of the antibody-drug conjugate shown in Formula I is 5-25 g / L or 10-20 g / L. In some embodiments of the pharmaceutical composition in liquid formulation, the concentration of the antibody-drug conjugate shown in Formula I is about 10 g / L or about 20 g / L.

[0125] In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the buffer (such as histidine-histidine hydrochloride buffer) is 1.1-11 g / L (e.g., about 1.1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 4.5 g / L, about 4.6 g / L, about 4.7 g / L, about 4.8 g / L, about 4.9 g / L, about 5 g / L, about 5.1 g / L, about 5.2 g / L, about 5.3 g / L, about 5.4 g / L, about 5.5 g / L, about 6 g / L, about 6.5 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, or about 11 g / L). 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 g / L. In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the buffer (such as histidine-histidine hydrochloride buffer) is about 3 g / L, about 3.02 g / L, about 4 g / L, or about 5 g / L.

[0126] In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is a histidine-histidine hydrochloride buffer, wherein the histidine content is 0.1-1 g / L (e.g., about 0.1 g / L, about 0.2 g / L, about 0.21 g / L, about 0.22 g / L, about 0.23 g / L, about 0.24 g / L, about 0.25 g / L, about 0.26 g / L, about 0.27 g / L, about 0. The concentrations are approximately 0.28 g / L, about 0.29 g / L, about 0.3 g / L, about 0.31 g / L, about 0.32 g / L, about 0.33 g / L, about 0.34 g / L, about 0.35 g / L, about 0.4 g / L, about 0.45 g / L, about 0.5 g / L, about 0.55 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, or about 1 g / L. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is a histidine-histidine hydrochloride buffer, wherein the histidine content is 0.1-0.7 g / L or 0.2-0.5 g / L. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is a histidine-histidine hydrochloride buffer, wherein the histidine content is about 0.34 g / L, about 0.4 g / L, or about 0.5 g / L.

[0127] In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is 1-10 g / L (e.g., about 1 g / L, about 2 g / L, about 2.5 g / L, about 2.6 g / L, about 2.7 g / L, about 2.8 g / L, about 2.9 g / L, about 3 g / L, about 3.5 g / L, about 3.6 g / L, about 4 g / L, about 4.5 g / L, about 4.6 g / L, about 4.7 g / L, about 4.8 g / L, about 4.9 g / L, about 5 g / L, about 5.1 g / L, about 5.2 g / L, about 5.3 g / L, about 5.4 g / L, about 5.5 g / L, about 6 g / L, about 6.5 g / L, about 7 g / L, about 8 g / L, about 9 g / L, or about 10 g / L). In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is 2-6 g / L or 2-4 g / L. In some embodiments of the pharmaceutical composition in liquid formulation, the buffer is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is about 2.68 g / L, about 3.6 g / L, or about 4.5 g / L.

[0128] 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 g / L (e.g., about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 81 g / L, about 82 g / L, about 83 g / L, about 84 g / L, about 85 g / L, about 86 g / L, about 87 g / L, about 88 g / L, about 89 g / L, about 90 g / L, about 91 g / L, about 92 g / L, about 93 g / L, about 94 g / L, about 95 g / L, about 100 g / L, about 105 g / L, or about 110 g / L). 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 g / L or 40-80 g / L. In some embodiments of the pharmaceutical composition in liquid formulation form, the content of the stabilizer (such as trehalose and / or sucrose) is about 40 g / L, about 60 g / L, or about 80 g / L.

[0129] In some embodiments of the pharmaceutical composition in liquid formulation form, the content of surfactant (such as polysorbate 20 and / or polysorbate 80) is 0.005-1 g / L (e.g., about 0.005 g / L, about 0.01 g / L, about 0.05 g / L, about 0.1 g / L, about 0.15 g / L, about 0.16 g / L, about 0.17 g / L, about 0.18 g / L, about 0.19 g / L, about 0.2 g / L, about 0.25 g / L, about 0.3 g / L, about 0.35 g / L, about 0.4 g / L, about 0.45 g / L, about 0.5 g / L, about 0.55 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, or about 1 g / L). In some embodiments of the pharmaceutical composition in liquid formulation, the content of the surfactant (such as polysorbate 20 and / or polysorbate 80) is 0.05-0.4 g / L. In some embodiments of the pharmaceutical composition in liquid formulation, the content of the surfactant (such as polysorbate 20 and / or polysorbate 80) is about 0.2 g / L.

[0130] In some embodiments of the pharmaceutical composition in liquid formulation form, the pharmaceutical composition comprises:

[0131] The antibody-drug conjugate shown in Formula I (e.g., any ADC molecule shown in Table 1) is present in a concentration of 1-30 g / L, 5-25 g / L, 10-20 g / L, about 10 g / L, or about 20 g / L.

[0132] Buffers (e.g., histidine-histidine hydrochloride buffers) in concentrations of 1.1-11 g / L, 2-6 g / L, about 3 g / L, about 3.02 g / L, about 4 g / L, or about 5 g / L;

[0133] Stabilizers (e.g., trehalose and / or sucrose) are present in concentrations of 30-110 g / L, 40-90 g / L, 40-80 g / L, approximately 40 g / L, approximately 60 g / L, or approximately 80 g / L; and

[0134] Surfactants (e.g., polysorbate 20 and / or polysorbate 80) are present in amounts of 0.005-1 g / L, 0.05-0.4 g / L, or about 0.2 g / L.

[0135] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, wherein the histidine content is 0.1-1 g / L or 0.1-0.7 g / L or 0.2-0.5 g / L or about 0.34 g / L or about 0.4 g / L or about 0.5 g / L, and the histidine hydrochloride content is 1-10 g / L or 2-6 g / L or 2-4 g / L or about 2.68 g / L or about 3.6 g / L or about 4.5 g / L.

[0136] In some embodiments of the pharmaceutical composition in liquid formulation, the pharmaceutical composition comprises about 20 g / L of an antibody-drug conjugate of Formula I (e.g., any ADC molecule shown in Table 1), about 0.34 g / L of histidine, about 2.68 g / L of histidine hydrochloride, about 80 g / L of trehalose and / or sucrose, and about 0.2 g / L of polysorbate 20 and / or polysorbate 80.

[0137] In some embodiments of the pharmaceutical composition in liquid formulation, the pharmaceutical composition comprises about 20 g / L of an antibody-drug conjugate of Formula I (e.g., any of the ADC molecules shown in Table 1), about 0.34 g / L of histidine, about 2.68 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, and about 80 g / L of sucrose, and the pH of the pharmaceutical composition in liquid formulation is about 5.3.

[0138] In some embodiments, the pharmaceutical composition comprises:

[0139] 1-30 or 5-25 or 10-20 or about 10 or about 20 parts by weight of the antibody-drug conjugate of Formula I (e.g., any of the ADC molecules shown in Table 1);

[0140] 1.1-11 or 2-6 or about 3.02 or about 4 or about 5 parts by weight of buffer (e.g., histidine-histidine hydrochloride buffer);

[0141] 30-110 or preferably 40-90 or 40-80 or about 40 or about 60 or about 80 parts by weight of stabilizer (such as trehalose and / or sucrose); and

[0142] 0.005-1 or 0.05-0.4 or about 0.2 parts by weight of surfactant (such as polysorbate 20 and / or polysorbate 80).

[0143] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, wherein the histidine is 0.1-1 or 0.1-0.7 or 0.2-0.5 or about 0.34 or about 0.4 or about 0.5 parts by weight, and the histidine hydrochloride is 1-10 or 2-6 or 2-4 or about 2.68 or about 3.6 or about 4.5 parts by weight.

[0144] In some embodiments, the pharmaceutical composition is a lyophilized injection, and when the lyophilized injection is diluted with water for injection, the diluted pharmaceutical composition contains about 20 g / L of an antibody-drug conjugate of Formula I, about 0.34 g / L of histidine, about 2.68 g / L of histidine hydrochloride, 0.2 g / L of polysorbate 80, 80 g / L of sucrose, and a pH of about 5.3.

[0145] In some embodiments, the pharmaceutical composition is a lyophilized injection, the stock solution of which comprises about 20 g / L of an antibody-drug conjugate of Formula I (e.g., any of the ADC molecules shown in Table 1), about 0.34 g / L of histidine, about 2.68 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, and about 80 g / L of sucrose, and the pH of the stock solution is about 5.3.

[0146] In some embodiments, the pharmaceutical composition is an injection containing about 20 g / L of an antibody-drug conjugate of Formula I (e.g., any of the ADC molecules shown in Table 1), about 0.34 g / L of histidine, about 2.68 g / L of histidine hydrochloride, about 0.2 g / L of polysorbate 80, and about 80 g / L of sucrose, and the pH of the injection is about 5.3.

[0147] In some embodiments, the pharmaceutical composition described in the first aspect is used to treat proliferative diseases. In some embodiments, the proliferative disease is selected from cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma). In some implementations, proliferative diseases are diseases associated with abnormal B7H3 expression, including cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

[0148] 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:

[0149] (1) Prepare the antibody-drug conjugate solution shown in Formula I;

[0150] (2) Preparation of ultrafiltration replacement buffer;

[0151] (3) Change the antibody-drug conjugate prepared in step (1) to the buffer solution prepared in step (2).

[0152] In some implementations, the method further includes (4) freeze drying.

[0153] In some implementations, step (2) includes weighing the buffer, stabilizer, and surfactant according to the prescribed amount, diluting them with water for injection to the target preparation volume, stirring and mixing them evenly to obtain an ultrafiltration replacement buffer.

[0154] In some implementations, step (3) includes using an ultrafiltration membrane to ultrafilter and replace the antibody-drug conjugate obtained in step (1) with an ultrafiltration replacement buffer prepared in step (2) by a certain volume, and diluting the antibody-drug conjugate to the target concentration to obtain the stock solution.

[0155] In some embodiments, step (2) may optionally include a step of sterile filtration of the resulting ultrafiltration replacement buffer.

[0156] In some implementation schemes, the freeze-drying step is performed under the following conditions:

[0157] (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;

[0158] (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 0.06mbar, time 300 minutes to 10000 minutes or 300 minutes to 4000 minutes or about 3000 minutes;

[0159] (3) The conditions for drying are: 15-45℃ or about 35℃ or about 30℃, pressure 0.1-0.5mbar (absolute pressure) or about 0.25mbar, time 90-5000 minutes or 500-2000 minutes or about 900 minutes.

[0160] 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. In some embodiments, the proliferative disease is selected from cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma). In some implementations, proliferative diseases are diseases associated with abnormal B7H3 expression, including cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

[0161] 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. In some embodiments, the proliferative disease is selected from cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma). In some implementations, proliferative diseases are diseases associated with abnormal B7H3 expression, including cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

[0162] In some implementations, cancers associated with abnormal B7H3 expression are cancers in which B7H3 (B7H3+) is highly expressed on the surface of cancer cells.

[0163] 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 expression of B7H3 (B7H3+) can be defined as at least 60% of cancer cells in a cancer cell population expressing B7H3, or at least 70% of cancer cells expressing B7H3, or at least 80% of cancer cells expressing B7H3, or at least 90% of cancer cells expressing B7H3, or at least 95% of cancer cells expressing B7H3, or at least 98% of cancer cells expressing B7H3, or at least 99% of cancer cells expressing B7H3.

[0164] In other embodiments, the degree of B7H3 positivity of the tumor can be analyzed qualitatively, quantitatively, or semi-quantitatively using various methods in the art.

[0165] The pharmaceutical composition of this application can be used alone or in combination with other antitumor agents.

[0166] 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. Long-term stability studies showed no significant changes in the physicochemical properties of the antibody portion, meeting the storage requirements for antibody preparations. The DAR value and DAR6 distribution of the ADC portion also showed no significant changes, indicating that the conjugate drug exhibits uniform quality during storage and can be stored for extended periods.

[0167] 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

[0168] 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.

[0169] 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.

[0170] 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.

[0171] The antibodies in this application can be prepared using recombinant DNA methods (e.g., U.S. Patent 4,816,567, etc.).

[0172] Common Procedure A: Purification of Antibody-Drug Conjugates

[0173] Equilibrate a 1 mL Mabselect SuRe pre-packed chromatography column on an AKTA system with PBS 7.0 / EDTA solution (10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0). Load the antibody-drug conjugate using a loading loop. After reequilibration with 10–20 mL of PBS 7.0 / EDTA, elute with acetate buffer at pH 3.5, and neutralize with 1 / 10 volume of sodium citrate neutralizing solution to obtain the purified antibody-drug conjugate.

[0174] Common Procedure B: Determination of the Antibody-Drug Conjugate Ratio (DAR) of Antibody-Drug Conjugates

[0175] Dithiothreitol was added to the antibody-drug conjugate to a final concentration of 20 mM, and the mixture was incubated in a water bath at 37°C for 30 minutes. This process cleaved the disulfide bonds between the antibody-drug conjugate chains, and the resulting sample was used for HPLC analysis. The HPLC system used was an Agilent Technologies 1260 Infinity HPLC, with a PLRP-S column (5 μm particle size; 2.1 mm × 50 mm; Agilent Technologies). The column temperature was 80°C. Mobile phase A was 0.1% trifluoroacetic acid (TFA) aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid (TFA) acetonitrile solution. The sample loading volume was 10 μL, and the gradient program was as follows: 0-3 min 27%-27%, 3-8 min 27%-35%, 8-25 min 35%-43%, 25-26 min 43%-95%, 26-31 min 95%-95%, 31-31.5 min 95%-27%, and 31.5-40 min 27%-27%. Hydrophobicity increased with the number of drugs linked to the drug chains (L0 and H0) compared to unlinked light chains (L1, linking one drug; H1, linking two drugs; H3, linking three drugs). Therefore, elution was performed in the order of L0, L1, H0, H1, H2, H3. The DAR value was calculated based on the peak area at 280 nm.

[0176] DAR=(L1 / (L0+L1)×1+H1 / (H0+H1+H2+H3)×1+H2 / (H0+H1+H2+H3)×2+H3 / (H0+H1+H2+H3)×3)×2.

[0177] Preparation Examples

[0178] Preparation Example 1: Preparation of Antibody-Drug Conjugate 43A-JSSW-4

[0179] Antibody reduction:

[0180] The 43A antibody medium prepared based on the sequence structure of the 43A antibody was replaced with PBS 7.0 / EDTA to prepare an antibody solution with a concentration of 10 mg / mL. This solution (1.0 mL) was placed into a 1.5 mL EP tube, and 15.64 μL of 10 mM TCEP (Tris(2-chloroethyl)phosphate, Bailingwei Technology Co., Ltd.) aqueous solution (equivalent to 2.3 equivalences per molecule of antibody) was added. The tube was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0181] Antibody-drug-adaptor compound conjugation:

[0182] Add 30.6 μL of 10 mM DMSO solution of JSSW-001 (Shanghai Haoyuan Biomedical Technology Co., Ltd.; equivalent to 4.5 antibody molecule) to the above solution at room temperature, mix well, and react at room temperature for 30 minutes to link the drug-adaptor compound to the antibody. Next, add 10.2 μL of 100 mM aqueous solution of N-ethylmaleimide (Bailingwei Technology Co., Ltd.; equivalent to 15 antibody molecule), and react at room temperature for 20 minutes to terminate the coupling reaction.

[0183] Purification of antibody-drug conjugates:

[0184] The above solution was purified by common operation A to obtain a solution containing the 43A-JSSW-4 conjugate.

[0185] Determination of the drug-antibody conjugate ratio (DAR) of antibody-drug conjugates:

[0186] DAR was determined using common operation B, and the average DAR value of the 43A-JSSW-4 conjugate was 3.8.

[0187] Preparation Example 2: Preparation of Antibody-Drug Conjugate 43A-JSSW-6

[0188] Antibody reduction:

[0189] The 43A antibody medium was replaced with PBS 7.0 / EDTA to prepare an antibody solution with a concentration of 10 mg / mL. This solution (1.0 mL) was placed into a 1.5 mL EP tube, and 10 mM TCEP aqueous solution (22.44 μL; equivalent to 3.3 equivalences per molecule of antibody) was added. The tube was incubated at 37 °C for 3 hours to reduce the disulfide bonds within the antibody.

[0190] Antibody-drug-adaptor compound conjugation:

[0191] Add 10 mM DMSO solution of JSSW-001 (43.5 μL; 6.4 equivalents per antibody molecule) to the above solution at room temperature, mix well, and react at room temperature for 30 minutes to link the drug-adaptor compound to the antibody. Next, add 100 mM aqueous solution of N-ethylmaleimide (10.2 μL; 15 equivalents per antibody molecule) and react at room temperature for 20 minutes to terminate the coupling reaction.

[0192] Purification of antibody-drug conjugates:

[0193] The above solution was purified by common operation A to obtain a solution containing the 43A-JSSW-6 conjugate.

[0194] Determination of the drug-antibody conjugate ratio (DAR) of antibody-drug conjugates:

[0195] DAR was measured using common operation B, and the average DAR value of the 43A-JSSW-6 conjugate was 5.8.

[0196] Preparation Example 3: Preparation of Antibody-Drug Conjugate 43A-JSSW-8

[0197] Antibody reduction:

[0198] The 43A antibody medium was replaced with PBS 7.0 / EDTA to prepare an antibody solution with a concentration of 10 mg / mL. This solution (1.0 mL) was placed into a 1.5 mL EP tube, and 100 mM TCEP aqueous solution (8.16 μL; equivalent to 12 equivalences per molecule of antibody) was added. The tube was incubated at 37 °C for 3 hours to reduce the disulfide bonds in the antibody.

[0199] Antibody-drug-adaptor compound conjugation:

[0200] Add 10 mM DMSO solution of JSSW-001 (68.0 μL; 10 equivalents per antibody molecule) to the above solution at room temperature, mix well, and react at room temperature for 30 minutes to link the drug-adaptor compound to the antibody. Next, add 100 mM aqueous solution of N-ethylmaleimide (10.2 μL; 15 equivalents per antibody molecule) and react at room temperature for 20 minutes to terminate the coupling reaction.

[0201] Purification of antibody-drug conjugates:

[0202] The above solution was purified by common operation A to obtain a solution containing the 43A-JSSW-8 conjugate.

[0203] Determination of the drug-antibody conjugate ratio (DAR) of antibody-drug conjugates:

[0204] DAR was measured using common operation B, and the average DAR value of the 43A-JSSW-8 conjugate was 7.5.

[0205] Preparation Example 4: Preparation of Antibody-Drug Conjugate 43B-JSSW-6

[0206] Antibody reduction:

[0207] The 43B antibody medium prepared based on the sequence structure of the 43B antibody was replaced with PBS 7.0 / EDTA to prepare an antibody solution with a concentration of 10 mg / mL. This solution (1.0 mL) was placed into a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (equivalent to 12 equivalents per molecule of antibody) was added. The tube was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0208] Antibody-drug-adaptor compound conjugation:

[0209] Add 10 mM DMSO solution of JSSW-001 (68.0 μL; 10 equivalents per antibody molecule) to the above solution at room temperature, mix well, and react at room temperature for 30 minutes to link the drug-adaptor compound to the antibody. Next, add 100 mM aqueous solution of N-ethylmaleimide (10.2 μL; 15 equivalents per antibody molecule) and react at room temperature for 20 minutes to terminate the coupling reaction.

[0210] Purification of antibody-drug conjugates:

[0211] The above solution was purified by common operation A to obtain a solution containing the 43B-JSSW-6 conjugate.

[0212] Determination of the drug-antibody conjugate ratio (DAR) of antibody-drug conjugates:

[0213] DAR was determined using common operation B, and the average DAR value of the 43B-JSSW-6 conjugate was 5.6.

[0214] Preparation of Control Example 1: Preparation of Antibody-Drug Conjugate M30-DXd-4

[0215] In the ADC control example, the antibody selected was antibody M30-H1-L4 (referred to as "M30" in this application) from CN103687945A. The antibody sequence is as follows, and the antibody moiety was prepared based on its structure:

[0216] M30 heavy chain (SEQ ID NO:12)

[0217] M30 light chain (SEQ ID NO:13)

[0218] The drug selected is Deruxtecan (Shanghai Haoyuan Biomedical Technology Co., Ltd.) (referred to as "DXd" in this application).

[0219] The structural schematic diagram of the ADC control example M30-DXd-4 is shown above, and the fabrication process is as follows.

[0220] Antibody reduction:

[0221] The laboratory-prepared M30 antibody medium was replaced with PBS 7.0 / EDTA to prepare an antibody solution with a concentration of 10 mg / mL. This solution (1.0 mL) was placed into a 1.5 mL EP tube, and 10 mM TCEP aqueous solution (15.64 μL; equivalent to 2.3 equivalences per molecule of antibody) was added. The tube was incubated at 37 °C for 3 hours to reduce the disulfide bonds within the antibody.

[0222] Antibody-drug-adaptor compound conjugation:

[0223] Add 30.6 μL of 10 mM Deruxtecan DMSO solution (equivalent to 4.5 antibody molecule) to the above solution at room temperature, mix well, and react at room temperature for 30 minutes to link the drug-adaptor compound to the antibody. Next, add 10.2 μL of 100 mM N-ethylmaleimide aqueous solution (equivalent to 15 antibody molecule) and react at room temperature for 20 minutes to terminate the coupling reaction.

[0224] Purification of antibody-drug conjugates:

[0225] The above solution was purified by common operation A to obtain a solution containing the M30-DXd-4 conjugate.

[0226] Determination of the drug-antibody conjugate ratio (DAR) of antibody-drug conjugates:

[0227] DAR was measured using common operation B, and the average DAR value of the M30-DXd-4 conjugate was 4.2.

[0228] Test Example 1: Biomembrane Interference Determination of Antibody Affinity

[0229] The affinity of anti-B7H3 antibodies 43A and 43B (prepared in the same manner as described in the above preparation examples) for human B7H3 (Beijing Biosciences Co., Ltd., catalog number: B7B-H52E7) was determined using a ForteBio Octet red 96e instrument (Sartorius). Different test samples at 5 μg / ml were immobilized using a Protein A capture sensor. The B7H3 antigen was serially diluted, starting at 100 nM and then 2-fold, for a total of 7 gradients. The buffer system was PBS pH 7.4 + 0.02% Tween 20. The steps were: ① Baseline 60s, ② Loading 800s (antibody immobilization, threshold set to 0.50 nm), ③ Baseline 2 120s, ④ Association 120s, ⑤ Dissociation 300s, ⑥ Regeneration 300s. Blank buffer was used for blanking, the baseline was aligned with the y-axis, and the data was fitted using Savitzky-Golay data analysis software in Octet. The affinity test results for antibodies 43A and 43B are shown in Table 2.

[0230] Table 2. Affinity of antibodies 43A and 43B to human B7H3

[0231] Test Example 2: Cell Binding Activity Assay

[0232] The affinity of anti-B7H3 antibodies 43A and 43B was detected using the B7H3-positive human melanoma cell line A375, with M30 antibody as a positive control. Each antibody was diluted to 15000 ng / mL with PBS containing 2% BSA, and then serially diluted 11 times to achieve antibody test sample concentrations ranging from 0.014 to 15000 ng / mL. A375 cells were centrifuged at 500 × g for 5 min, washed three times with PBS containing 2% BSA, and incubated with samples of different dilutions at 4°C for 2 h. After washing, the cells were incubated with goat anti-human Alexa Fluor 488 fluorescent dye at 4°C in the dark for 1 h. After washing and resuspending, the mean fluorescence intensity (MFI) of the cells was detected using an Attune NxT flow cytometer (Thermo Fisher Scientific, inc.).

[0233] Experimental results showed that both antibodies 43A and 43B could bind with high affinity to B7H3-positive A375 cells, EC 50 The concentrations were 54.5 ng / ml and 61.2 ng / ml, respectively, both significantly better than the EC of M30. 50 125.3 ng / ml.

[0234] Test Example 3: Endocytosis Activity Study

[0235] Antibodies M30, 43A, and 43B were incubated with human melanoma cells A375 at 4°C for 2 hours. After incubation, the cells were washed twice with PBS, culture medium was added, and then the cells were incubated at 37°C in a 5% CO2 incubator for endocytosis. Cells were harvested after the specified incubation time. The cells were then stained with goat anti-human Alexa Fluor 488 and incubated at 4°C in the dark for 60 minutes. After incubation, the cells were washed twice with experimental buffer (2% FBS / PBS), resuspended in buffer, and then analyzed using flow cytometry. The median fluorescence signal of the corresponding wells in the experimental plate was read using a plate reader.

[0236] Internalization rate % = (initial average median fluorescence value - average median fluorescence value at different time points) / initial average median fluorescence value × 100%.

[0237] The experimental results are shown in Table 3. After antibodies 43A and 43B bind to A375 cells, they can be rapidly internalized into the cells, and the internalization rate is faster than that of M30.

[0238] Table 3. Internalization rate of antibodies at different time points for each antibody-drug conjugate.

[0239] Test Example 4: Inhibitory effect of antibody-drug conjugate on the in vitro growth of human melanoma A375 cells

[0240] A375 cells were collected, resuspended as single-cell suspensions, and cell viability and counts were determined using trypan blue staining. The cell density was adjusted to 1 × 10⁻⁶ cells / cells. 5 Cells / mL; 100 μL per well was added to a 96-well black flat-bottom cell culture plate; 20 μL of serially diluted antibody-drug conjugate was added to each well of the pre-inoculated 96-well black flat-bottom cell culture plate; the plate was incubated in a cell culture incubator (37℃, 5% CO2) for 168±8 hours; 20 μL of 0.03% sodium resazurin solution was added to each well; the plate was incubated at 37℃ for 3-4 hours, and the fluorescence values ​​were read at 550 nm / 610 nm using a microplate reader. The half-inhibitory concentration (IC50) of the test sample was calculated using Prism or similar plotting software. 50 .

[0241] In in vitro experiments on human melanoma cells A375, 43A-JSSW-4, 43A-JSSW-6, 43A-JSSW-8, and 43B-JSSW-6 all exhibited good cell growth inhibitory activity. The results are shown in Table 4.

[0242] Table 4. In vitro growth inhibition activities of various antibody-drug conjugates

[0243] Test Example 5: Anti-tumor experiment of antibody-drug conjugate against human melanoma cell line A375 mouse xenografts

[0244] In this embodiment, age-appropriate female NU / NU mice were inoculated with human melanoma cells A375 until the tumor volume reached approximately 100-200 mm. 3 At that time, 28 animals with good tumor growth were selected and divided into 4 groups according to tumor volume. The animal grouping and drug administration / dosage regimen are shown in Table 5.

[0245] Table 5 Animal grouping and dosage regimen

[0246] After grouping, the mice were administered the drug and weighed. Data were recorded. The tumor diameter was measured at different time points after drug administration to dynamically observe tumor growth and calculate tumor volume using the following formula:

[0247] Tumor volume (mm) 3 = 1 / 2 × major diameter (mm) × [minor diameter (mm)] 2

[0248] Tumor inhibition rate TGI% = [1-(Ti-T0) / (Ci-C0)]×100%, where T0 and C0 are the average 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 average tumor volumes at the end of the experiment for the drug administration group and the solvent control group, respectively.

[0249] At the experimental endpoint, compared with the solvent group, all antibody-drug conjugates significantly inhibited tumor growth. The tumor inhibition rates of 43A-JSSW-4, 43A-JSSW-6 and 43A-JSSW-8 were 53.6%, 65.2% and 87.7%, respectively. Detailed information is shown in Table 6.

[0250] Table 6. Efficacy of various antibody-drug conjugates against A375 xenografts in tumor-bearing mice.

[0251] Test Example 6: Antitumor Experiment of Antibody-Drug Conjugate against Calu-6 Mouse Xenografts of Human Lung Cancer Cells

[0252] In this embodiment, age-appropriate female NOD / SCID mice were inoculated with human lung cancer cells Calu-6 until the tumor volume reached approximately 100-200 mm. 3 Thirty-five animals with good tumor growth were selected and divided into five groups according to tumor volume. The grouping and administration / dosage regimens are shown in Table 7.

[0253] Table 7 Animal grouping and dosage regimen

[0254] After grouping, the mice were administered the drug and weighed. Data were recorded. The tumor diameter was measured at different time points after drug administration to dynamically observe tumor growth and calculate tumor volume using the following formula:

[0255] Tumor volume (mm) 3 = 1 / 2 × major diameter (mm) × [minor diameter (mm)] 2

[0256] Tumor inhibition rate TGI% = [1-(Ti-T0) / (Ci-C0)]×100%, where T0 and C0 are the average 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 average tumor volumes at the end of the experiment for the drug administration group and the solvent control group, respectively.

[0257] At the experimental endpoint, all antibody-drug conjugates significantly inhibited tumor growth compared to the solvent group; detailed information is shown in Table 8.

[0258] Table 8. Efficacy of various antibody-drug conjugates against Calu-6 xenografts in tumor-bearing mice.

[0259] Test Example 7: Antitumor Experiment of Antibody-Drug Conjugate against Human Esophageal Squamous Cell Carcinoma Xenografts in KYSE-150 Mice

[0260] In this embodiment, age-appropriate female NU / NU mice were inoculated with human esophageal squamous cell carcinoma cells KYSE-150 until the tumor volume reached approximately 100 mm. 3 Around 18 days after the tumors started growing, 18 animals with good tumor growth were selected and grouped equally according to tumor volume. The animal grouping and drug administration / dosage regimen are shown in Table 9.

[0261] Table 9 Animal grouping and dosage regimen

[0262] After grouping, the mice were administered the drug and weighed. Data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after drug administration, and tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = 1 / 2 × major diameter (mm) × [minor diameter (mm)] 2

[0263] Tumor inhibition rate TGI% = [1-(Ti-T0) / (Ci-C0)]×100%, where T0 and C0 are the average 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 average tumor volumes at the end of the experiment for the drug administration group and the solvent control group, respectively.

[0264] At the experimental endpoint, compared with the solvent group, 43B-JSSW-6 significantly inhibited tumor growth, while no anti-cancer effect was observed in M30-DXd-4. Detailed information is shown in Table 10.

[0265] Table 10. Efficacy of various antibody-drug conjugates against KYSE-150 xenografts in tumor-bearing mice.

[0266] Test Example 8: Antitumor Experiment of Antibody-Drug Conjugate against Human Non-Small Cell Lung Cancer SK-MES-1 Mouse Xenograft

[0267] In this embodiment, age-appropriate female NU / NU mice were inoculated with human non-small cell lung cancer cells SK-MES-1 until the tumor volume reached approximately 100 mm. 3 Around 18 days after the tumors started growing, 18 animals with good tumor growth were selected and grouped equally according to tumor volume. The animal grouping and drug administration / dosage regimen are shown in Table 11.

[0268] Table 11 Animal grouping and dosage regimen

[0269] After grouping, the mice were administered the drug and weighed. Data were recorded. The tumor diameter was measured at different time points after drug administration to dynamically observe tumor growth and calculate tumor volume using the following formula:

[0270] Tumor volume (mm) 3 = 1 / 2 × major diameter (mm) × [minor diameter (mm)] 2

[0271] Tumor inhibition rate TGI% = [1-(Ti-T0) / (Ci-C0)]×100%, where T0 and C0 are the average 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 average tumor volumes at the end of the experiment for the drug administration group and the solvent control group, respectively.

[0272] At the experimental endpoint, compared with the solvent group, 43B-JSSW-6 significantly inhibited tumor growth. M30-DXd-4 also showed anti-cancer effects, but weaker than 43B-JSSW-6. Detailed information is shown in Table 12.

[0273] Table 12. Efficacy of various antibody-drug conjugates against SK-MES-1 xenografts in tumor-bearing mice.

[0274] Test Example 9: Antitumor Experiment of Antibody-Drug Conjugate against Hep3B Mouse Xenografts of Human Hepatocellular Carcinoma

[0275] In this embodiment, age-appropriate female NU / NU mice were inoculated with human hepatocellular carcinoma cells Hep3B until the tumor volume reached approximately 100-200 mm. 3 At that time, 12 animals with good tumor growth were selected and the animals were divided into groups according to tumor volume. The grouping of animals and the administration / dosage regimen are shown in Table 13.

[0276] Table 13 Animal grouping and dosage regimen

[0277] After grouping, the mice were administered the drug and weighed. Data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after drug administration, and tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = 1 / 2 × major diameter (mm) × [minor diameter (mm)] 2

[0278] Tumor inhibition rate TGI% = [1-(Ti-T0) / (Ci-C0)]×100%, where T0 and C0 are the average 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 average tumor volumes at the end of the experiment for the drug administration group and the solvent control group, respectively.

[0279] At the experimental endpoint, 43B-JSSW-6 significantly inhibited tumor growth compared to the solvent group. Detailed information is shown in Table 14.

[0280] Table 14. Efficacy of antibody-drug conjugates against Hep3B xenografts in tumor-bearing mice.

[0281] Test Example 10: Antitumor Experiment of Antibody-Drug Conjugate against Human Lung Cancer Calu-6 Mouse Xenografts

[0282] In this embodiment, age-appropriate NOD / SCID mice were inoculated with human lung cancer cells Calu-6 until the tumor volume reached approximately 100-200 mm. 3 Eighteen animals with good tumor growth were selected and grouped equally according to tumor volume. The animal grouping and drug / dosage regimen are shown in Table 15.

[0283] Table 15 Animal grouping and dosage regimen

[0284] After grouping, the mice were administered the drug and weighed. Data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after drug administration, and tumor volume was calculated using the following formula: Tumor volume (mm²) 3 = 1 / 2 × major diameter (mm) × [minor diameter (mm)] 2

[0285] Tumor inhibition rate TGI% = [1-(Ti-T0) / (Ci-C0)]×100%, where T0 and C0 are the average 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 average tumor volumes at the end of the experiment for the drug administration group and the solvent control group, respectively.

[0286] At the experimental endpoint, compared with the solvent group, both antibody-drug conjugates significantly inhibited tumor growth, and 43B-JSSW-6 was significantly more effective than the control M30-DXd-4. Detailed information is shown in Table 16.

[0287] Table 16. Efficacy of various antibody-drug conjugates against Calu-6 xenografts in tumor-bearing mice.

[0288] Formulation Examples

[0289] The following examples use 43B-JSSW-6 of this application as an example to test and evaluate the effects of different formulation components and parameters on ADC formulations.

[0290] Formulation Example 1: pH and buffer system screening.

[0291] 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.

[0292] 20 mM histidine-histidine hydrochloride buffer (“H” group), citrate-sodium citrate buffer (“C” group), and succinate-sodium hydroxide buffer (“S” group) with pH values ​​of 5.0, 5.5, 6.0, and 6.5 were prepared. 43B-JSSW-6 was ultrafiltered into these 12 buffers to achieve a final protein concentration of approximately 10 mg / ml. After filtration through a 0.22 μm microporous membrane, stability tests were performed under high temperature (40±2℃) and light exposure (white light: 5000±500 Lux, UV: 90 μW / cm²). The particle size distribution of 43B-JSSW-6 under different pH values ​​and buffer conditions was determined using DLS, and the change in monomer purity of 43B-JSSW-6 under different pH values ​​and buffer conditions was determined using SEC-HPLC.

[0293] 1. Changes in appearance and particle size

[0294] Table 17 shows the changes in appearance and particle size of antibody-drug conjugates during high-temperature and light exposure tests. At T0 and 7 days of high temperature, all 12 test groups were colorless and clear solutions. At 5 days of light exposure, some test groups showed significant changes in appearance, with the pH 6.0 and 6.5 sodium citrate test groups and the pH 6.5 succinic acid test group exhibiting turbidity. Particle size data at T0 showed that, at the same pH, the histidine-histidine hydrochloride test group had significantly smaller particle size than the other two buffer test groups. At 7 days of high temperature, the pH 5.0 citrate-sodium citrate test group showed the largest change in particle size, while the succinic acid-sodium hydroxide test group showed a slight increase. The histidine-histidine hydrochloride test group showed no significant change. At 5 days of light exposure, the pH 5.0 and 5.5 histidine-histidine hydrochloride test groups showed no significant change in particle size, while the particle size of other test groups increased slightly, with the pH 6.5 succinic acid-sodium hydroxide test group showing a significant increase to 12.3 nm. The pH 5.0-5.5 histidine-histidine hydrochloride buffer is the preferred buffer solution.

[0295] Table 17 Results of the effects of high temperature and light on appearance and particle size Note: H: Histidine - Histidine hydrochloride, C: Citric acid - Sodium citrate, S: Succinic acid - Sodium hydroxide

[0296] 2. SEC Changes

[0297] 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 (LMWS) peaks was calculated according to the area normalization method.

[0298] Table 18 shows the effects of high temperature and light exposure on the purity of SEC-HPLC monomers. After 7 days of high temperature exposure, at the same pH, the histidine-histidine hydrochloride group showed the highest SEC monomer purity. Under the same buffer salt conditions, the SEC monomer purity was relatively high in the pH 5.0 and 5.5 groups. After 5 days of light exposure, at the same pH, the histidine-histidine hydrochloride group showed the highest SEC monomer purity. Under the same buffer solution, the purity of SEC monomers decreased with increasing pH. A pH of 5.0-5.5 with histidine-histidine hydrochloride was the preferred buffer solution.

[0299] Table 18 Results of the effects of high temperature and light on the purity of SEC-HPLC monomers Note: H: Histidine - Histidine hydrochloride, C: Citric acid - Sodium citrate, S: Succinic acid - Sodium hydroxide

[0300] Formulation Example 2: Screening of pH and Buffer Salt Concentration

[0301] 15mM, 20mM, and 25mM histidine-histidine hydrochloride buffers at pH 5.0, 5.3, and 5.5 were prepared. 43B-JSSW-6 was ultrafiltered into these nine buffers, resulting in a final protein concentration of approximately 10 mg / ml. After filtration through a 0.22 μm microporous membrane, stability tests were conducted at high temperature (40±2℃) for 7 days and under light exposure (white light: 5000±500 Lux, UV: 90 μW / cm²) for 5 days. The particle size distribution of 43B-JSSW-6 at different pH values ​​and buffer salt concentrations was determined using DLS, and the changes in the content of free toxins and small molecule toxins at different pH values ​​and buffer salt concentrations were determined using HPLC-MS.

[0302] 1. Particle size variation

[0303] The particle size of the 15 mM histidine-histidine hydrochloride system samples was smaller than that of the 20 mM and 25 mM histidine-histidine hydrochloride systems at the same pH. The particle size tended to increase with increasing buffer salt concentration. High temperature and light did not affect the particle size.

[0304] Table 19 Results of the effects of high temperature and light on particle size Note: H: Histidine-Histidine hydrochloride

[0305] 2. Changes in free toxin content

[0306] After 5 days of light exposure, the free toxin content of the 9 samples showed significant differences. At the same pH, the free toxin content increased with increasing buffer salt concentration, with the 20mM and 25mM groups exhibiting 2-3 times higher free toxin content than the 15mM histidine-histidine hydrochloride group. After 7 days of high temperature exposure, the free toxin content decreased with increasing pH at the same buffer concentration. Based on the combined analysis of particle size and free toxin content results, 15mM histidine-histidine hydrochloride at pH 5.3 was selected as the optimal buffer system.

[0307] Table 20 Results of the effects of high temperature and light on free toxin content

[0308] Formulation Example 3: Screening of Protein Content and Excipient Concentration

[0309] 15mM histidine-histidine hydrochloride buffer solutions at pH 5.3 containing 4%, 6%, and 8% sucrose were prepared. Samples containing 4%, 6%, and 8% sucrose were obtained by ultrafiltration. 10 mg / ml and 20 mg / ml 43B-JSSW-6 samples containing 4%, 6%, and 8% sucrose were then prepared. After filtration through a 0.22 μm microporous membrane, the samples were subjected to high temperature (45±2℃) for 5 days and light exposure for 5 days (white light: 5000±500 Lux, UV: 90 μW / cm²). 2Stability tests were conducted. The thermal stability of 43B-JSSW-6 at different sucrose concentrations was determined using DSC, and the changes in free toxin content in 43B-JSSW-6 samples at different protein and sucrose concentrations were determined using HPLC-MS.

[0310] 1. Changes in thermal stability

[0311] The initial sample was diluted to 1 mg / ml with the corresponding buffer solution. The thermal stability of the diluted sample was detected by DSC. The initial thermal denaturation temperatures (TOnset) of 43B-JSSW-6 were 49.50℃ (4% sucrose concentration), 50.05℃ (6% sucrose concentration), and 50.62℃ (8% sucrose concentration). TOnset increased with increasing sucrose concentration.

[0312] 2. Changes in free toxin content

[0313] Table 21 shows the effects of high temperature and light on free toxins in samples with different protein contents and sucrose concentrations. The free toxin content did not change significantly after the protein concentration increased from 10 mg / mL to 20 mg / mL. Considering the convenience of subsequent freeze drying and use, 8% sucrose and a protein concentration of 20 mg / mL were preferred.

[0314] Table 21 Results of the effects of high temperature and light on free toxin content

[0315] In addition, in terms of appearance, freeze-dried powder without added sucrose will have a honeycomb-like shape, which will affect the reconstitution effect during use.

[0316] Formulation Example 4: Lyophilization Process Development

[0317] Using 20 mg / ml antibody-drug conjugate 43B-JSSW-6, 0.34 g / L histidine, 2.68 g / L histidine hydrochloride, 0.2 g / L polysorbate 80, and 80 g / L sucrose as the base formulation, the buffer, surfactant, and stabilizer were weighed and diluted with water for injection to the target preparation volume. The mixture was stirred and stirred until homogeneous to obtain the ultrafiltration replacement buffer. The prepared antibody-drug conjugate was then transferred to the ultrafiltration replacement buffer.

[0318] 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 -29.58℃, and lyophilization microscopy showed that the disintegration temperature was -27.4℃.

[0319] Pre-freezing: Based on the glass transition temperature (-29.58℃) determined by the 43B-JSSW-6 stock solution, the pre-freezing temperature was set at -40℃. Before reaching -40℃, the plates were first brought to 5℃ over 30 minutes and maintained at 5℃ for another 30 minutes to ensure that all samples could be cooled from 5℃. The cooling rate was 0.5℃ / min, and the holding time was 180 minutes.

[0320] Single-stage drying: After pre-freezing, the sample enters the single-stage drying process, where over 90% of the water in the product is removed through sublimation. To improve the sublimation rate, while ensuring that the temperature of the unsublimated portion does not exceed its disintegration temperature, the plate temperature is set above the product's allowable disintegration temperature (-5℃). The vacuum setting is changed to conduct a single-stage drying study. Increasing the vacuum degree of the single-stage drying shortens the drying time, but results in higher residual moisture. Therefore, the proposed single-stage drying temperature is -5℃, and the vacuum degree is 0.06 mbar.

[0321] Table 22 Parameters and Moisture Data for Primary Drying Process Development

[0322] Desorption drying: After the first drying cycle, based on the product's thermal denaturation temperature, desorption drying is carried out at 35°C with a vacuum degree of 0.20 mbar.

[0323] A comparative study was conducted on the key mass molecular size isomers (SEC), charge variants (CEX), DAR values, and free toxin content of the solutions before and after lyophilization of the lyophilized samples. The results showed that the product quality did not change significantly after lyophilization, and the lyophilization process did not have a significant impact on the product quality.

[0324] Table 23 Comparison of mass between the solution before lyophilization and the reconstituted solution after lyophilization

[0325] Formulation Example 5: Bioactivity Assay

[0326] Using the reconstituted aqueous solution of the 43B-JSSW-6 formulation obtained in Formulation Example 4, 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.

[0327] Formulation Example 6: Stability Study

[0328] Two batches of lyophilized samples were prepared using the formulation and lyophilization process in Formulation Example 4. After lyophilization, the samples were subjected to long-term stability studies at 2-8°C. The appearance, moisture content, purity, free toxins, and bioactivity of the samples were tested using the relevant analytical methods described in the aforementioned examples. The product quality was stable, and no significant changes were observed in any of the key indicators, indicating that the formulation and lyophilization process of this application are robust and can produce products with stable quality.

[0329] Table 24 Results of stability studies on lyophilized formulations "LOD" represents the detection limit of the analytical method.

[0330] SEC-HPLC purity is used to separate ADC monomers from aggregates and fragments based on molecular size under non-denaturing conditions. The sum of peaks eluted before the main peak is reported as aggregates, and the sum of peaks eluted after the main peak is reported as fragments.

[0331] NR-CE-SDS purity is determined by separating the light chain, heavy chain, and other degradation products of ADC based on molecular size under non-reducing conditions. Due to the coupling mechanism of ADC, the inter-chain disulfide bonds are broken, resulting in free light chain, heavy chain, and other separated peaks. The sum of light chain and heavy chain peaks and the sum of other peaks are reported.

[0332] CZE purity represents the percentage of the main peak in CZE. The charge heterogeneity of the ADC is evaluated by measuring the percentage of acidic and basic variants using capillary zone electrophoresis.

[0333] The DAR distribution represents the number of toxins conjugated to the ADC molecule, assesses the number of conjugation sites and the percentage of the maximum conjugation number DAR6, and also counts the amount of antibodies that did not undergo conjugation.

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate of Formula I: Where Ab is an antibody or its antigen-binding fragment targeting B7 homology 3 (B7H3). R is selected from C 1-6 Alkyl, preferably C 1-3 Alkyl, more preferably methyl, ethyl, propyl or isopropyl, n is an integer or decimal in the range of 1-8, 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 injection, further preferably an injection 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 structure shown in formula Ia or formula Ib:

3. The pharmaceutical composition of claim 1, wherein the antibody-drug conjugate of formula I has the structure shown in formula I-1, Ia-1, or Ib-1:

4. The pharmaceutical composition according to any one of claims 1-3, wherein: Ab 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 light chain 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. The CDRs are determined according to the Kabat numbering rules. Preferably, the heavy chain variable region (VH) sequence is as shown in SEQ ID NO:7, and the light chain variable region (VL) sequence is as shown in SEQ ID NO:

8. More preferably, the antibody targeting B7H3 comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:9 or 10, and a light chain with an amino acid sequence as shown in SEQ ID NO:

11.

5. The pharmaceutical composition according to any one of claims 1-4, wherein Ab is a humanized antibody or an antigen-binding fragment thereof.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the antibody-drug conjugate of formula I has the following structure: Where d is an integer or decimal in the range of 2-8, preferably 2, 4, 6 or 8, and in, 43A is an antibody targeting B7H3 comprising two identical heavy and light chains, the heavy chain amino acid sequence of which is shown in SEQ ID NO:9 and the light chain amino acid sequence of which is shown in SEQ ID NO:

11. 43B is an antibody targeting B7H3 comprising two identical heavy and light chains, the heavy chain amino acid sequence of which is shown in SEQ ID NO:10 and the light chain amino acid sequence of which is shown in SEQ ID NO:

11.

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

8. The pharmaceutical composition according to any one of claims 1-7, 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%, 10-25%, or 10-20%, for example, about 19.38%.

9. The pharmaceutical composition according to any one of claims 1-8, 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.

10. The pharmaceutical composition of claim 9, wherein the buffer content is 1.01-12% by mass, preferably 2-6% or 3-5%, for example, about 3%, about 4%, about 5% or about 2.93%, based on 100 wt% of the pharmaceutical composition.

11. The pharmaceutical composition of claim 9 or 10, 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% or 0.3-0.5%, for example about 0.3%, about 0.4%, about 0.5% or about 0.33%, and the mass percentage of histidine hydrochloride is 1-10%, preferably 1.8-5.4% or 2.5-4.5%, for example about 2.5%, about 3.5%, about 4.5% or about 2.60%.

12. The pharmaceutical composition according to any one of claims 1-11, 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), more preferably sugars, and even more preferably sucrose and / or trehalose.

13. The pharmaceutical composition of claim 12, wherein the stabilizer comprises 1-90% by mass, preferably 30%-90% or 35-80%, for example about 39%, about 40%, about 50%, about 60%, about 70%, about 80% or about 77.50%, based on 100 wt% of the pharmaceutical composition.

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

15. The pharmaceutical composition of claim 14, wherein the surfactant is present in a mass percentage of 0.01-1%, preferably 0.05-0.4%, for example, about 0.1%, about 0.2%, about 0.3%, or about 0.19%, based on 100 wt% of the pharmaceutical composition.

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 6) has a mass percentage of 1-30%, preferably 5-25% or 10-25% or 10-20%, for example about 19.38%. A buffer having a mass percentage of 1-12%, preferably 2-6% or 3-5%, for example, about 3%, about 4%, about 5% or about 2.93% (for example, the buffer is a histidine-histidine hydrochloride buffer, preferably, the histidine mass percentage is 0.01-2%, preferably 0.25-0.75%, 0.2-0.6% or 0.3-0.5%, for example, about 0.3%, about 0.4%, about 0.5% or about 0.33%, and the histidine hydrochloride mass percentage is 1-10%, preferably 1.8-5.4% or 2.5-4.5%, for example, about 2.5%, about 3.5%, about 4.5% or about 2.60%); Stabilizers (e.g., trehalose and / or sucrose) at a mass percentage of 1-90%, preferably 30%-90% or 35-80%, such as about 39%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 77.50%; and The surfactant (e.g., polysorbate 20 and / or polysorbate 80) is present in a mass percentage of 0.01-1%, preferably 0.05-0.4%, for example about 0.1%, about 0.2%, about 0.3% or about 0.19%; Optionally, the pharmaceutical composition contains residual moisture.

17. The pharmaceutical composition of any one of claims 1-16, 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 6) has a mass percentage of about 19.38%. The buffer has a mass percentage of about 2.93% (for example, the buffer is a histidine-histidine hydrochloride buffer, wherein the mass percentage of histidine is about 0.33% and the mass percentage of histidine hydrochloride is about 2.60%). Stabilizers (e.g., trehalose and / or sucrose), at a mass percentage of approximately 77.50%; and Surfactants (e.g., polysorbate 20 and / or polysorbate 80) are present in a mass percentage of approximately 0.19%. Optionally, the pharmaceutical composition contains residual moisture.

18. The pharmaceutical composition of any one of claims 1-6, 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.

19. The pharmaceutical composition according to any one of claims 1-6, wherein the pharmaceutical composition is a liquid formulation, preferably an injection.

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

21. The pharmaceutical composition according to any one of claims 18-20, 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.

22. The pharmaceutical composition of claim 21, wherein the buffer (e.g., histidine-histidine hydrochloride buffer) is present in a concentration of 1.1-11 g / L, preferably 2-6 g / L, for example about 3 g / L, about 3.02 g / L, about 4 g / L, or about 5 g / L.

23. The pharmaceutical composition of claim 21 or 22, wherein the buffer is a histidine-histidine hydrochloride buffer, the histidine content is 0.1-1 g / L, preferably 0.1-0.7 g / L or 0.2-0.5 g / L, for example about 0.34 g / L, about 0.4 g / L or about 0.5 g / L, and the histidine hydrochloride content is 1-10 g / L, preferably 2-6 g / L or 2-4 g / L, for example about 2.68 g / L, about 3.6 g / L or about 4.5 g / L.

24. The pharmaceutical composition according to any one of claims 18-23, 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.

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

26. The pharmaceutical composition of any one of claims 18-25, wherein the solution, stock solution and / or liquid formulation further comprises a surfactant; preferably, the surfactant is selected from polysorbates, more preferably polysorbate 20 and / or polysorbate 80.

27. The pharmaceutical composition of claim 26, wherein the surfactant (e.g., polysorbate 20 and / or polysorbate 80) is present in an amount of 0.005-1 g / L, preferably 0.05-0.4 g / L, for example about 0.2 g / L.

28. The pharmaceutical composition of any one of claims 18-27, 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, and even more preferably 5.0-5.5, for example, about 5.

3.

29. The pharmaceutical composition of any one of claims 18-28, 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 6) has a content of 1-30 g / L, preferably 5-25 g / L or 10-20 g / L, for example about 10 g / L or about 20 g / L; A buffer, with a content of 1.1-11 g / L, preferably 2-6 g / L, for example about 3 g / L, about 3.02 g / L, about 4 g / L or about 5 g / L (for example, the buffer is a histidine-histidine hydrochloride buffer, preferably, the histidine content is 0.1-1 g / L, preferably 0.1-0.7 g / L or 0.2-0.5 g / L, for example about 0.34 g / L, about 0.4 g / L or about 0.5 g / L, and the histidine hydrochloride content is 1-10 g / L, preferably 2-6 g / L or 2-4 g / L, for example about 2.68 g / L, about 3.6 g / L or about 4.5 g / L); Stabilizers (e.g., trehalose and / or sucrose) are present in a concentration of 30-110 g / L, preferably 40-90 g / L or 40-80 g / L, for example, about 40 g / L, about 60 g / L or about 80 g / L; and The surfactant (e.g., polysorbate 20 and / or polysorbate 80) is present in a concentration of 0.005-1 g / L, preferably 0.05-0.4 g / L, for example about 0.2 g / L.

30. The pharmaceutical composition of any one of claims 18-29, wherein the solution, stock solution and / or liquid formulation comprises about 20 g / L of an antibody-drug conjugate of formula I (e.g., any antibody-drug conjugate of claim 6), about 0.34 g / L of histidine, about 2.68 g / L of histidine hydrochloride, about 80 g / L of trehalose and / or sucrose, and about 0.2 g / L of polysorbate 20 and / or polysorbate 80.

31. The pharmaceutical composition of any one of claims 18-30, wherein the solution, stock solution, and / or liquid formulation comprises: 1-30 (preferably 5-25 or 10-20, for example about 10 or about 20) parts by weight of the antibody-drug conjugate of Formula I (e.g., any antibody-drug conjugate of claim 6); 1.1-11 (preferably 2-6, e.g., about 3.02, about 4, or about 5) parts by weight of a buffer (e.g., the buffer is a histidine-histidine hydrochloride buffer, preferably, histidine is 0.1-1 (preferably 0.1-0.7 or 0.2-0.5, e.g., about 0.34, about 0.4, or about 0.5) parts by weight, and histidine hydrochloride is 1-10 (preferably 2-6 or 2-4, e.g., about 2.68, about 3.6, or about 4.5) parts by weight); 30-110 (preferably 40-90 or 40-80, e.g., about 40, about 60 or about 80) 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 20 and / or polysorbate 80).

32. The pharmaceutical composition according to any one of claims 1-31, for treating proliferative diseases, preferably, said proliferative disease is a disease related to abnormal expression of B7H3, including cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

33. A method for preparing the pharmaceutical composition according to any one of claims 1-31, comprising the following steps: (1) Prepare the antibody-drug conjugate solution 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, stabilizer, and surfactant according to the prescription amount, diluting them 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) at a certain volume, diluting the antibody-drug conjugate to the target concentration to obtain the stock solution; Optionally, the method further includes a step of sterile filtration of the obtained ultrafiltration replacement buffer; and further optionally, the method further includes lyophilizing the stock solution, preferably, lyophilization comprising: (1) Pre-freezing, (2) One-time drying, and (3) Desorption drying; More 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), for example about 0.06 mbar, and time 300 minutes to 10,000 minutes, preferably 300 minutes to 4,000 minutes, for example about 3,000 minutes; (3) The conditions for drying are: 15-45°C, for example about 35°C or about 30°C, pressure 0.1-0.5mbar (absolute pressure), for example about 0.25mbar, time 90-5000 minutes, preferably 500-2000 minutes, for example about 900 minutes.

34. Use of the pharmaceutical composition of any one of claims 1-31 in the preparation of a medicament for treating proliferative diseases, preferably, said proliferative disease is a disease related to abnormal expression of B7H3, including cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

35. A method for treating or preventing proliferative diseases, the method comprising administering to a patient in need of a therapeutically effective dose of a pharmaceutical composition of any one of claims 1-31; preferably, the proliferative disease is a disease associated with abnormal expression of B7H3, including cancers such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, esophageal squamous cell carcinoma, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

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