Drug composition of recombinant anti-human CLDN18.2 monoclonal antibody-MMAE conjugate

Abstract

The present application provides a pharmaceutical composition comprising an anti-CLDN18.2 antibody-drug conjugate, a buffer, a stabilizer and a surfactant. The present application also provides a method for preparing the pharmaceutical composition and uses of the pharmaceutical composition.

Description

Technical Field

[0001] Citation of Related Applications This application claims the priority of a Chinese patent application filed on May 31, 2022, with the invention title "Drug Composition of Recombinant Anti-Human CLDN18.2 Monoclonal Antibody-MMAE Conjugate" and the application number 202210606878.4. The entire content of this patent application is incorporated herein by reference for all purposes.

[0002] Technical Field This application generally relates to the field of biopharmaceuticals, and specifically relates to a drug composition of an antibody-drug conjugate, a method for preparing the drug composition, and the use of the drug composition.

Background Art

[0003] Claudin is a protein family that widely exists in vertebrate epithelial and endothelial cells and plays a role in constructing an intercellular barrier to control the flow of intercellular molecules. CLDN18.2 (Claudin 18.2) is one of the most studied members of the Claudin protein family. It is usually expressed only in gastric epithelial cells and has high tissue specificity. When cancer occurs, Claudin18.2 is highly expressed in various tumor tissues such as gastric cancer (60% - 80%), pancreatic cancer (50%), esophageal cancer (30% - 50%), and lung cancer (40% - 60%). The Claudin18.2 protein is usually embedded in the cell membrane of epithelial cells, but Claudin18.2 on the surface of tumor cells is exposed due to the destruction of tight junctions by malignant tumors and becomes an attackable target. Thus, Claudin 18.2 has become a new target for targeted therapy and immunotherapy with great potential.

[0004] Gastric cancer ranks third in cancer - related mortality and is considered one of the most difficult - to - cure cancers worldwide. In patients with advanced or metastatic gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, the median overall survival (mOS) is less than 10 months. Since the number of patients with Claudin 18.2 targets accounts for about 50% - 60% of all gastric cancer patients compared to human epidermal growth factor receptor 2 (HER2) targets, treatment targeting this target is extremely promising. Currently, product types targeting Claudin 18.2 worldwide cover various types such as monoclonal antibodies, bispecific antibodies, CAR - T, and ADC, including the current main product forms.

[0005] In this field, anti - cancer products targeting Claudin 18.2 are strongly in demand.

Summary of the Invention

[0006] In a first aspect, the present application provides a pharmaceutical composition comprising an anti - CLDN18.2 antibody - drug conjugate, a buffer, a stabilizer, and a surfactant, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises HCDR1 - HCDR3 having the amino acid sequences shown in SEQ ID NO:1, 2, and 3 respectively, and / or the light chain comprises LCDR1 - LCDR3 having the amino acid sequences shown in SEQ ID NO:4, 5, and 6 respectively, and the structure of the drug & linker part (L&D) of the antibody - drug conjugate is as follows.

Chemical formula

[0007] In a third aspect, the present application provides a method for treating cancer, comprising administering to a cancer - affected individual in need an effective amount of the pharmaceutical composition described in the first aspect.

[0008] In a fourth aspect, the present application provides a method for preparing the pharmaceutical composition described in the first aspect, comprising the following steps.

[0009] The step of preparing the anti-CLDN18.2 antibody-drug conjugate, The step of solution replacement of the anti-CLDN18.2 antibody-drug conjugate into a liquid system containing other components of the drug composition, and The steps of sterile filtration and aseptic filling.

Brief Description of the Drawings

[0010]

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Modes for Carrying Out the Invention

[0011] Definitions Unless otherwise defined, all scientific terms used in this specification have the same meaning as understood by those skilled in the art. For the definitions and terms in this field, those skilled in the art can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes that refer to one of the 20 common L-amino acids used in this field.

[0012] The broad scope of the present application shows numerical ranges and approximate values of parameters. However, the numerical values shown in specific embodiments are described as accurately as possible. Nevertheless, any numerical value inherently necessarily includes a certain error, which is due to the standard deviation existing in each measurement. Also, all ranges disclosed in this specification should be understood to cover any and all sub-ranges included therein. For example, the described range of "1 to 10" should be understood to include any and all sub-ranges between the minimum value of 1 and the maximum value of 10 (including the endpoints), that is, all sub-ranges starting from a value greater than or equal to the minimum value of 1 (e.g., 1 to 6.1), and all sub-ranges ending at a value less than or equal to the maximum value of 10 (e.g., 5.5 to 10). It should be understood that the references "incorporated herein" are all incorporated in their entirety.

[0013] As used herein, the term "antibody-drug conjugate (ADC)", also referred to as "antibody-drug complex", refers to a drug having cytotoxicity conjugated to an antibody. Some ADCs targeting cancer cells can selectively deliver the drug to cancer cells because the antigen targeted by the antibody can be expressed on the surface of cancer cells and the antibody also binds to an antigen that can be internalized by cells, thereby causing the accumulation of the drug in cancer cells and killing the cancer cells.

[0014] As used herein, the term "drug composition" refers to a combination of at least one drug and any pharmaceutically acceptable carrier or excipient combined to achieve a specific purpose. Pharmaceutically acceptable carriers include water, buffered aqueous solutions, isotonic saline solutions (e.g., PBS (phosphate buffered saline)), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol or polyalkylene glycols (e.g., polypropylene glycol), triglycerides, etc. The type of pharmaceutically acceptable carrier used is determined in particular by whether the composition according to the present application is prepared for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present application may contain, as additives, wetting agents, emulsifying agents or buffering substances.

[0015] The drug composition according to the present application can be administered by any suitable route, for example, oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.

[0016] As used herein, "therapeutically effective amount" or "effective amount" refers to an amount sufficient to show benefit to the subject being administered. The actual dosage, dosing rate and dosing period depend on the situation and severity of the treating individual. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of the general practitioner and other physicians, and is usually determined taking into account the disease being treated, the individual situation of the patient, the delivery site, the administration method, and other factors known to the physician.

[0017] As used herein, the terms "subject" or "individual" mean a mammal such as a human, but may also be other animals such as wild animals (e.g., herons, cranes, swans, etc.), domestic animals (e.g., ducks, geese, etc.) or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, groundhogs, chipmunks, etc.).

[0018] The term "antibody" broadly includes intact antibodies and any antigen-binding fragment ("antigen-binding portion") or single-chain form thereof. "Full-length / intact antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises one heavy-chain variable region (abbreviated as VH) and one heavy-chain constant region, and the heavy-chain constant region comprises three domains called CH1, CH2, and CH3. Each light chain comprises one light-chain variable region (abbreviated as VL) and one light-chain constant region, and the light-chain constant region comprises one domain called CL. The VH and VL regions can be further subdivided into multiple regions with high variability called complementarity-determining regions (CDRs), and multiple more conserved regions called framework regions (FRs) are interspersed therebetween. Each VH and VL consists of three CDRs and four FRs, and are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus towards the carboxyl terminus. These variable regions in the heavy and light chains comprise the binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Chimeric or humanized antibodies are also included in the antibodies according to the present application. The full-length / intact antibody may be any type of antibody, e.g., IgD, IgE, IgG, IgA, or IgM (or subclasses of the above antibodies), but is not limited to any particular class. Immunoglobulins can be specified into different classes by the antibody amino acid sequences of the heavy-chain constant regions. Immunoglobulins typically have five main classes, namely IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions corresponding to each class of immunoglobulins are called α, β, ε, γ, and μ, respectively. The subunit structure and three-dimensional structure of each class of immunoglobulins are known.

[0019] Complementary determining regions (CDRs, generally CDR1, CDR2, and CDR3) are the regions within the variable regions that most affect the affinity and specificity of an antibody. There are multiple common definition methods for the CDR sequences of VH or VL, including the IMGT, Chothia definition, and Kabat definition. In the embodiments of the present application, the CDR sequences in the VH and VL sequences can be determined according to the Kabat definition.

[0020] The term "humanized antibody" refers to an antibody that may contain CDR regions derived from human-derived antibodies, and other parts of the antibody molecule are derived from one (or more) human antibodies. Also, in order to maintain the binding affinity, some residues of the framework (referred to as FR) segments can be modified, and the humanized antibody or its fragment according to the present application can be prepared by techniques known to those skilled in the art.

[0021] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species. For example, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. The chimeric antibody or its fragment according to the present application can be prepared using genetic recombination techniques. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter, a sequence encoding the variable region of a non-human, particularly a mouse monoclonal antibody according to the present application, and a sequence encoding the human antibody constant region. The chimeric antibody according to the present application encoded by such a recombinant gene is, for example, a mouse-human chimeric antibody in which the specificity of the antibody is determined by the variable region derived from mouse DNA and the isotype is determined by the constant region derived from human DNA. For methods of preparing chimeric antibodies, reference can be made to, for example, the literature Verhoeyn et al. (Bio Essys, 8:74, 1988).

[0022] The term "monoclonal antibody" refers to a preparation of antibody molecules consisting of a single molecule. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0023] Based on the structural information of the anti-CLDN18.2 monoclonal antibody provided in the present application, it can be obtained by preparing with CHO-K1 (ATCC Number: CCL-61, Lot No.: 59965043) cells using methods known in the art.

[0024] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0025] In the present specification, the terms "transglutaminase" and "TGase" are used interchangeably and refer to an enzyme capable of carrying out a transglutamination reaction. The term "transglutamination" used in the present specification refers to a reaction in which the γ-glutamyl of a receptor glutamine residue derived from a protein / peptide is transferred to an amine group such as a primary amine or the ε-amino group of lysine.

[0026] The inventors of the present invention have conducted research and exploration on the pharmaceutics of antibody-drug conjugates / complexes (ADCs) and developed each invention of the present application.

[0027] In the research of antibody formulations, the formation of aggregates and the generation of degradation products cause pharmaceutically undesirable side effects, leading to an increase in immunogenicity or risks related to intravenous diseases in patients receiving drug treatment. For the above reasons, when preparing antibodies, it is necessary to suppress the formation of aggregates and the generation of degradation products. In view of this, researchers are considering the formulation forms and formulation prescriptions of various drug compositions (for example, aqueous injection or lyophilized injection forms). Similarly, when researching the drug formulations of antibody-drug conjugates, more technical problems are faced, and it is necessary to consider not only the specific properties of the antibody part but also the specific properties of the drug-linker part. For example, in the storage process of antibody-drug conjugates, small molecule toxins may fall off, and such shedding affects the effectiveness and toxicity of related drugs.

[0028] One of the technical objectives of the present application is to provide a drug composition (especially in the form of an aqueous injection) of an antibody-drug conjugate and a related preparation method that can suppress the formation of aggregates and the generation of degradation products and reduce the shedding of toxins.

[0029] As an example, the inventors specifically modified glutamine at a specific site (Q295) in the heavy chain of a recombinantly developed anti-human CLDN18.2 monoclonal antibody by means of catalysis by microbial transglutaminase (mTgase), and complexed it with a small molecule microtubule inhibitor drug MMAE to obtain a related antibody-drug conjugate, the average drug-antibody ratio (DAR) of which is 2.0, among which the distribution of DAR2 exceeds 70%, and the product uniformity is high. Based on this, a liquid formulation prescription particularly suitable for the above antibody-drug conjugate was prepared. The realized technical advantages include at least one of significantly reducing production costs, shortening the production stage, making administration more convenient, and avoiding the disadvantages such as the complex reconstitution program when using freeze-dried drugs and the high requirements for the technical level of the preparer, on the premise of meeting the stability requirements.

[0030] In a first aspect, the present application provides a drug composition comprising an antibody-drug conjugate, a buffer, a stabilizer, and a surfactant.

[0031] In some embodiments, the structure of the drug & linker moiety (L&D) in the antibody-drug conjugate is shown in the following formula I.

[0032]

Chemical formula

[0033] In some embodiments, the antibody comprises a heavy chain and a light chain, the heavy chain comprises HCDR1-HCDR3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and / or the light chain comprises LCDR1-LCDR3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain and a light chain having the amino acid sequences shown in SEQ ID NOs: 9 and SEQ ID NO: 10, respectively. In some embodiments, the antibody is a monoclonal antibody or a bispecific antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a fully humanized antibody. In some specific embodiments, the antibody has ADCC activity. In some specific embodiments, the antibody has CDC activity. In some specific embodiments, the antibody specifically binds to CLDN18.2 and does not substantially bind to CLDN18.1. In some specific embodiments, the antibody is an IgG1κ antibody.

[0034] In some embodiments, the buffer comprises, but is not limited to, acetate, succinate, gluconate, histidine salt, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0035] In some embodiments, the histidine salt buffer is a buffer containing histidine ions. Examples of histidine salt buffers include buffers such as histidine - histidine hydrochloride, histidine - acetate, histidine - phosphate, histidine - sulfate, etc., where the histidine - acetate buffer is prepared from histidine and acetic acid, and the histidine - histidine hydrochloride buffer is prepared from histidine and histidine hydrochloride. Similarly, the succinate buffer may be succinic acid - sodium succinate, and the citrate buffer may be citric acid - sodium citrate.

[0036] In some embodiments, the buffer is a histidine - histidine hydrochloride buffer.

[0037] In some embodiments, the stabilizer includes, but is not limited to, saccharides (e.g., sucrose, trehalose), polyhydric alcohols (e.g., mannitol, sorbitol), amino acids (L - serine, sodium glutamate, alanine, glycine, sarcosine, etc.).

[0038] In some embodiments, the surfactant includes, but is not limited to, polysorbate 20 or polysorbate 80.

[0039] In some embodiments, the pH of the drug composition is 5.0 - 7.4. In some embodiments, the pH of the drug composition is 5.0 - 6.5. In some embodiments, the pH of the drug composition is 5.5.

[0040] In some embodiments, the drug composition further contains water.

[0041] In some embodiments, the components of the drug composition include the following alone or in common.

[0042] (a) Histidine-histidine hydrochloride with a content of 5 - 30 mM, for example, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM (b) Polysorbate 20 or polysorbate 80 with a content of 0.02 - 0.08 wt%, for example, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt% (c) Sucrose with a content of 6 - 8 wt%, for example, about 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt% (d) Antibody-drug conjugate with a content of 5 - 50 mg / ml, for example, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml (e) pH 5.0 - 6.5, for example, about 5.0, about 5.5, about 6.0, about 6.5 In some embodiments, the pharmaceutical composition comprises the following.

[0043] (a) 5 - 30 mM of histidine-histidine hydrochloride (b) 0.02 - 0.08 wt% of polysorbate 20 or polysorbate 80 (c) 6 - 8 wt% of sucrose (d) 5 - 50 mg / ml of antibody-drug conjugate, and (e) pH 5.0 - 6.5 In some embodiments, the pharmaceutical composition comprises the following.

[0044] (a) 10 - 30 mM of histidine-histidine hydrochloride (b) 0.02 - 0.04 wt% of polysorbate 20 or polysorbate 80 (c) 6 to 7 wt% sucrose (d) 10 to 20 mg / ml antibody-drug conjugate, and (e) pH 5.0 - 6.0 In some embodiments, the pharmaceutical composition comprises the following.

[0045] (a) 20 mM histidine - histidine hydrochloride (b) 0.02 wt% polysorbate 20 (c) 6 wt% sucrose (d) 10 mg / ml antibody-drug conjugate, and (e) pH 5.5 In some embodiments, the pharmaceutical composition can be prepared as a lyophilized formulation, a liquid formulation, or a powder for injection.

[0046] In some embodiments, the pharmaceutical composition of the present application is used for the treatment of cancer. In some embodiments, the cancer is CLDN18.2 positive cancer.

[0047] In some embodiments, the cancer is a cancer that expresses CLDN18.2 on the surface of cancer cells. In some embodiments, the cancer is a cancer that highly expresses CLDN18.2 (CLDN18.2+). In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) refers to the fact that at least 60% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) refers to the fact that at least 70% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) refers to the fact that at least 80% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) refers to the fact that at least 90% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) refers to the fact that at least 95% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) refers to the fact that at least 98% of the cancer cells in the cancer cell population express CLDN18.2. In some embodiments, a cancer that highly expresses CLDN18.2 (CLDN18.2+) refers to the fact that at least 99% of the cancer cells in the cancer cell population express CLDN18.2.

[0048] In some embodiments, the pharmaceutical composition of the present application can be used for the treatment of at least one cancer selected from breast cancer, gastric cancer (also referred to as gastric adenocarcinoma), colorectal cancer (also referred to as colon and rectal cancer, including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, endometrial cancer, uterine cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, multiple myeloma, neuroepithelial tissue tumor, schwannoma, head and neck cancer, skin cancer, laryngeal cancer, gallbladder cancer, cholangiocarcinoma, mesothelioma and sarcoma.

[0049] In some embodiments, the pharmaceutical composition of the present application can be used for the treatment of at least one cancer selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer and uterine carcinosarcoma.

[0050] In some embodiments, the pharmaceutical composition of the present application can be used for the treatment of at least one cancer selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma and Paget's disease.

[0051] In some embodiments, the pharmaceutical composition of the present application can be used for the treatment of breast cancer, gastric cancer, colorectal cancer or non-small cell lung cancer.

[0052] In some embodiments, since the pharmaceutical composition of the present application can be selectively used as an agent for drug therapy, which is a main method for treating cancer, it can delay the growth of cancer cells, suppress their proliferation, and further kill cancer cells. Due to these effects, it is possible to eliminate the symptoms caused by cancer in cancer patients, improve the QOL (quality of life score) of cancer patients, and maintain the lives of cancer patients to obtain a therapeutic effect. Even if the pharmaceutical composition and treatment method of the present application do not result in killing cancer cells, by suppressing or controlling the proliferation of cancer cells, it is possible to achieve a higher QOL (quality of life score) for cancer patients and realize a longer survival while pursuing a longer survival.

[0053] In some embodiments, the pharmaceutical composition of the present application may be used alone or in combination with other therapies, for example, in combination with surgery, radiotherapy, hormone therapy, etc.

[0054] In some embodiments, the pharmaceutical composition of the present application can be administered in combination with other cancer therapeutic agents to enhance the anti-cancer effect. Examples of other cancer therapeutic agents used for such purposes include, but are not limited to, 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT 11), docetaxel, pemetrexed, sorafenib, vinblastine, vinorelbine, everolimus, tanespimycin, bevacizumab, oxaliplatin, lapatinib, and any agent having anti-tumor activity.

[0055] In a second aspect, the present application provides the use of the pharmaceutical composition described in the first aspect in the preparation of a drug for treating cancer.

[0056] In a third aspect, the present application provides a method for treating cancer, which includes administering an effective amount of the pharmaceutical composition described in the first aspect to a cancer-afflicted individual in need thereof.

[0057] The embodiments and technical features regarding the medical use of the pharmaceutical composition of the present application in the first aspect are also applicable to the second and third aspects.

[0058] In the fourth aspect, the present application provides a method for preparing the pharmaceutical composition described in the first aspect, which includes the following steps.

[0059] The step of preparing the anti-CLDN18.2 antibody-drug conjugate, The step of liquid displacement of the anti-CLDN18.2 antibody-drug conjugate into a liquid system containing other components of the pharmaceutical composition, and The steps of sterile filtration and aseptic filling.

[0060] In some specific embodiments, the method for preparing the pharmaceutical composition of the present application includes the following steps. (1) The step of preparing an aqueous solution containing a predetermined amount of the following substances (i) Antibody-drug conjugate, (ii) Histidine buffer, (iii) Sucrose, and (iv) Surfactant (2) The step of adjusting the pH of the aqueous solution to a predetermined value as needed (3) The step of dispensing into injection bottles after sterile filtration

[0061] In some embodiments, the method for preparing the pharmaceutical composition of the present application includes the following steps. (1) Liquid displacement of the antibody-drug conjugate by ultrafiltration: Prepare a histidine-histidine hydrochloride solution to a concentration equivalent to the formulation amount of the final product, use a 30kD ultrafiltration membrane pack, and set the replacement ratio of liquid displacement by ultrafiltration to 100 times or more. Control the pH of the replacement solution to be consistent with that of the final product, and perform liquid displacement so that the concentration of the antibody-drug conjugate is equal to or higher than (optimally 1.2 times or more) the concentration of the final product. Replace the original affinity chromatography solution with the histidine-histidine hydrochloride solution.

[0062] (2) Preparation of semi-finished product dilution: Based on the formulation amount of this product and the drug solution concentration of the antibody-drug conjugate after liquid replacement by ultrafiltration, calculate the preparation amount of the final product, calculate the required amount of each auxiliary material, and prepare a semi-finished product dilution solution. Add about 80% of water for injection to the mixing container, weigh histidine, histidine hydrochloride, polysorbate 20, and sucrose in sequence and add them to the mixing container, stir and mix. After each auxiliary material is completely dissolved, add water for injection to make up the volume to the preparation amount, stir and mix uniformly, and then filter for use preparation.

[0063] (3) Manufacture of finished product: Add the prepared semi-finished product dilution to the preparation solution containing the antibody-drug conjugate drug solution that has been liquid-replaced by ultrafiltration, make up the volume to the preparation amount of the semi-finished product with the dilution, stir and mix uniformly, sample to detect the protein content, pH value and microbial limit, after sterile filtration, dispense into sterile injection bottles according to the product specifications, stopper and cap.

[0064] In a fifth aspect, the present application further provides a lyophilized preparation prepared by lyophilizing the liquid drug composition prepared in the fourth aspect. In some embodiments, the preparation method of the lyophilized preparation includes the steps of pre-freezing, vacuum suction, primary drying and finishing drying.

[0065] Examples Hereinafter, the present application will be further described with reference to specific examples. It should be understood that these examples are only for explaining the present application and do not limit the scope of the present application. In the following examples, for experimental methods where specific conditions are not specified, generally, normal conditions or conditions recommended by the manufacturer are followed.

[0066] Example 1: Preparation and efficacy test of anti-CLDN18.2 antibody-drug conjugate 1.1 Preparation of anti-CLDN18.2 antibody-drug conjugate A certain volume of LND1002 (the structure is shown in Figure 2), reaction buffer, CLDN18.2 monoclonal antibody (the exemplary antibody structural sequences employed in this example can be found in Table 30. Using conventional technical means in this field, such as genetic engineering techniques, vectors containing heavy-chain and light-chain related sequences are expressed in host cells and obtained by purification.), mTGase (transglutaminase), H 2 O were each introduced into an elastic ethylene-vinyl acetate disposable reaction bag by a peristaltic pump in an appropriate order. After sealing the reaction bag and mixing uniformly, it was placed at 30°C for a reaction time of 24 - 144 h was set. During the reaction process, sampling was carried out every 24 h, and the conjugation rate was detected by C4-HPLC analysis. When the conjugation rate ≥ 95%, the reaction was terminated and purified immediately to obtain the antibody-drug conjugate SYJS001. Its overall structure is shown in Figure 2, and the antibody-related sequences can be found in Table 30. Specifically, the heavy chain of the antibody contains HCDR1 - HCDR3 with the amino acid sequences shown in SEQ ID NO: 1, 2, and 3 respectively, the light chain of the antibody contains LCDR1 - LCDR3 with the amino acid sequences shown in SEQ ID NO: 4, 5, and 6 respectively, the heavy chain of the antibody contains the heavy-chain variable region with the amino acid sequence shown in SEQ ID NO: 7, and the light chain of the antibody contains the light-chain variable region with the amino acid sequence shown in SEQ ID NO: 8.

[0067] The amino acid sequence of the transglutaminase used in this example is as follows.

[0068] DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP(SEQ ID NO:19)

[0069] 1.2 Detection of the binding ability between SYJS001-ADC and CLDN18.2 protein by flow cytometry In this experiment, the binding ability between SYJS001-ADC and CLDN18.2 proteins of different species was detected by flow cytometry.

[0070] Cell lines overexpressing CLDN18.2 (HEK293-human CLDN18.2, HEK293-mouse CLDN18.2, CHO-K1-cynomolgus monkey CLDN18.2) were incubated with SYJS001-ADC samples at different concentrations, and then incubated with a secondary antibody that binds to IgG (goat anti-human IgG(H+L) cross-adsorbed secondary antibody). The fluorescence signal values at different concentrations were detected by a flow cytometer, and the binding ability of the samples to CLDN18.2 of different species was analyzed.

[0071] Specific experimental steps: The starting concentration of the protein sample SYJS001-ADC was 45 μg / ml, and it was diluted in a 3-fold gradient, with a total of 11 gradients. 100 μl of antibody dilutions at different concentrations were taken and added to cells expressing human, mouse, and cynomolgus monkey CLDN18.2 (1×10 6Incubate at 4°C for 1.5 h with (cells / ml, 100 μl / well), wash the cells to remove unbound samples, add goat anti-human IgG (H+L) cross-adsorbed secondary antibody (diluted 1:1000), incubate at 4°C for 1 hour, after washing, detect the fluorescence signal value of the corresponding well using a flow cytometer, analyze the data with GraphPad Prism 5 software, select the regression model of the four-parameter equation to create an "S" curve, and the software automatically generates the median effective dose ED 50 (C value). As shown in Figure 3 and Table 1, the experimental results indicate that SYJS001-ADC has good affinity for human, mouse, and cynomolgus monkey CLDN18.2.

[0072]

Table 1

[0073] 1.3 Specific binding of SYJS001-ADC to CLDN18.2 HEK293-human CLDN18.1 cells and CHOK1-human CLDN18.2 cells were prepared in the same manner as in Example 1.2.

[0074] In the experiment to detect the binding specificity of the SYJS001 monoclonal antibody (Figure 4), HEK293-human CLDN18.1 cells and CHOK1-human CLDN18.2 cells were cultured in the corresponding complete medium, subcultured every 2 - 3 days, and when the cells reached 90% confluence, the cell density was adjusted to 2 - 3×10 6Adjust to cells / mL, and seed the cell suspension into a 96-well plate for flow cytometry at 100 μL / well using a multi-channel pipette. Centrifuge at 2500 rpm for 5 min, discard the supernatant, wash twice with 2% FBS / PBS, dilute the SYJS001 monoclonal antibody with 2% FBS / PBS buffer respectively, set up two multi-wells for each test substance concentration, and also set up corresponding blank controls. Add 100 μL per well, incubate at 4°C for 2 hours, wash three times with 2% FBS / PBS, add 488-labeled sheep anti-human IgG (diluted at 1:1000), incubate at 4°C for 1 hour, wash three times with 2% FBS / PBS, and then select the corresponding fluorescence channel for reading.

[0075] In the experiment to detect the binding specificity between the SYJS001 monoclonal antibody and SYJS001-ADC (Figure 5), culture HEK293-human CLDN18.2 cells (Example 1.2) in the corresponding complete medium, passage every 2 - 3 days, and when the cells reach 90% confluence, adjust the cell density to 2 - 3×10 6 cells / mL, seed the cell suspension into a 96-well plate for flow cytometry at 100 μL / well using a multi-lane pipette, centrifuge at 2500 rpm for 5 min, discard the supernatant, wash twice with 2% FBS / PBS, dilute the SYJS001 monoclonal antibody and SYJS001-ADC with 2% FBS / PBS buffer respectively. The starting concentration of the ADC is 15 μg / ml, and it is diluted in a 3-fold gradient for a total of 11 gradients. Set up two multi-wells for each test substance concentration, and also set up corresponding blank controls. Add 100 μL per well, incubate at 4°C for 2 hours, wash three times with 2% FBS / PBS, add 488-labeled sheep anti-human IgG (diluted at 1:1000), incubate at 4°C for 1 hour, wash three times with 2% FBS / PBS, and then select the corresponding fluorescence channel for reading.

[0076] As a result (see Figures 4 and 5), the monoclonal antibody used in SYJS001 obtained in the present application can specifically bind to CLDN18.2 and has no obvious cross-reaction with CLDN18.1. Compared with the naked antibody, SYJS001-ADC can also specifically bind to CLDN18.2, and it is shown that the complexation of the toxin has no significant effect on the affinity.

[0077] 1.4 In vivo efficacy evaluation of SYJS001-ADC 1.4.1 Efficacy comparison with gemcitabine and cisplatin as controls 1) In this experiment, a human pancreatic cancer Bxpc3-18.2 nude mouse xenograft tumor model was selected. When the tumor volume grew to about 100 mm 3 (on the 39th day after inoculation), 48 animals with well-growing tumors were selected. The animals were evenly divided into 6 groups (day 0) of 8 animals each according to tumor volume. They were respectively administered 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), SYJS001-ADC at 2, 4, and 8 mg / kg (single administration), SYJS001-mAb (SYJS001 naked antibody) at 8 mg / kg (single administration), and gemcitabine (GEM) at 50 mg / kg (biw×4, twice a week for 4 weeks) by intravenous injection. The tumor diameter was measured twice a week, the body weight of the mice was weighed, the data were recorded, and the growth status of the tumors was dynamically observed by measuring the tumor diameter at different time points after administration. The experiment was terminated on the 28th day. After the mice were asphyxiated with carbon dioxide, the tumors were dissected and the tumor weights were weighed.

[0078] As a result, in this experiment, the tumor weight inhibition rates in the SYJS001-ADC 2, 4, and 8 mg / kg (single-dose) groups, the SYJS001-mAb 8 mg / kg (single-dose) group, and the Gemcitabine 50 mg / kg (biw×4) group were shown to be 56.6%, 94.8%, 97.8%, -36.2%, and 51.0% respectively. Compared with the solvent control group (0.9% sodium chloride injection), the SYJS001-ADC 2, 4, 8 mg / kg (single-dose) groups and the GEM 50 mg / kg (biw×4: administered twice a week for a total of 4 doses) group were all able to significantly inhibit tumor growth (P<0.01). Compared with the positive control GEM 50 mg / kg (biw×4) group, the SYJS001-ADC 4 and 8 mg / kg (single-dose) groups had a more significant inhibitory effect on tumors. Especially in the 8 mg / kg group, the inhibitory effect was nearly twice as high (p<0.001) (see Figure 6).

[0079] 2) In this experiment, human gastric cancer NUGC-4-18.2 was selected to construct a nude mouse transplanted tumor model. When the tumor volume reached approximately 120 mm 3 (on the 6th day after inoculation), 64 animals with well-growing tumors were selected. The animals were evenly divided into 8 groups of 8 animals each (day 0) according to tumor volume, and were intravenously administered 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), SYJS001-ADC 1, 2, 4 mg / kg (qw×3), 4, 8 mg / kg (single-dose), SYJS001-mAb 4 mg / kg (qw×3), and Cisplatin 6 mg / kg (qw×3, once a week for a total of 3 weeks). The tumor diameter was measured twice a week, the body weight of the mice was weighed, the data were recorded, and the growth changes of the tumors were dynamically observed by measuring the tumor diameter at different time points after administration. The experiment was terminated on the 20th day. After the animals were asphyxiated with carbon dioxide, the tumors were dissected and the tumor weights were weighed.

[0080] As a result, in this experiment, the tumor weight inhibition rates in the SYJS001-ADC 1, 2, 4 mg / kg (qw×3) groups, 4, 8 mg / kg (single administration) groups, SYJS001-mAb 4 mg / kg (qw×3) group, and cisplatin 6 mg / kg (qw×3) group were shown to be 98.0%, 100%, 100%, 100%, 100%, -1.2%, and 66.5% respectively. Compared with the solvent control group, each group except the SYJS001-mAb 4 mg / kg (qw×3) group could significantly inhibit tumor growth (p<0.001). The SYJS001-ADC 1, 2, 4 mg / kg (qw×3), 4, 8 mg / kg (single administration) groups were significantly superior to the positive control group of cisplatin 6 mg / kg (qw×3) in terms of both efficacy and toxicity (p<0.05) (see Figure 7).

[0081] 1.4.2 Efficacy comparison with IMAB362-ADC as a control IMAB362 (also known as zolbetuximab), a previously reported IgG1 subtype chimeric monoclonal antibody, selectively targets the first extracellular domain of CLDN18.2 and has extremely low activity against CLDN18.1, so it was selected as the control antibody in this experiment.

[0082] 1) In this experiment, a human pancreatic cancer Bxpc3-18.2 nude mouse transplanted tumor model was selected, and when the tumor volume was approximately 100 mm 3On the 39th day after inoculation, when the tumors had grown, 64 animals with well-growing tumors were selected. The animals were evenly divided into 8 groups of 8 each (day 0) according to tumor volume, and were intravenously administered with 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), SYJS001-ADC at 2, 4, and 8 mg / kg (single dose), IMAB362-ADC (for the IMAB362 sequence, refer to Patent CN201680021997.6. Gene synthesis was completed by Nanjing GeneScript Biotechnology Co., Ltd., protein expression was transiently transfected and expressed by the KOP293 transient transfection protein expression system of Zhuhai Kairui Biotechnology Co., Ltd., and the preparation of IMAB362-ADC refers to the preparation method of SYJS001-ADC in Example 1.1.) at 2, 4, and 8 mg / kg (single dose), and SYJS001-mAb at 8 mg / kg (single dose). The tumor diameter was measured twice a week, the body weight of the mice was weighed, the data were recorded, and the tumor growth status was dynamically observed by measuring the tumor diameter at different time points after administration. The experiment was terminated on the 28th day. After the mice were asphyxiated with carbon dioxide, the tumors were dissected and the tumor weights were weighed.

[0083] As a result, in this experiment, it was shown that the tumor volumes of the SYJS001-ADC 2, 4, and 8 mg / kg (single dose) groups, the SYJS001-mAb 8 mg / kg (single dose) group, and the IMAB362-ADC 2, 4, and 8 mg / kg (single dose) groups were 35.9%, 6.0%, 3.4%, 116.2%, 47.0%, 6.8%, and 5.1% of the solvent control group, respectively. Compared with the solvent control group, both the SYJS001-ADC 2, 4, and 8 mg / kg (single dose) groups and the IMAB362-ADC 2, 4, and 8 mg / kg (single dose) groups could significantly inhibit tumor growth (p<0.01). Compared with the positive control IMAB362-ADC group, the difference in the effect at high doses was not large, but under low-dose (effective dose) conditions, the in vivo tumor inhibitory effect of SYJS001-ADC was significantly superior to that of IMAB362-ADC. Specifically, refer to Figure 8 (RTV (relative tumor volume), relative tumor volume).

[0084] 2) In this experiment, human gastric cancer NUGC-4-18.2 was selected to construct a nude mouse transplanted tumor model. When the tumor volume reached approximately 120 mm 3 (on the 6th day after inoculation), 64 animals with well-growing tumors were selected. The animals were evenly divided into 8 groups of 8 each (day 0) according to tumor volume, and were intravenously administered with 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), SYJS001-ADC at 0.5, 1, 2, 4 mg / kg (single administration), IMAB362-ADC at 0.5, 1, 2, 4 mg / kg (single administration), and SYJS001-mAb at 4 mg / kg (single administration). The tumor diameter was measured twice a week, the body weight of the mice was weighed, the data were recorded, and the growth changes of the tumors were dynamically observed by measuring the tumor diameter at different time points after administration. The experiment was terminated on the 20th day. After the animals were asphyxiated with carbon dioxide, the tumors were dissected and the tumor weights were weighed.

[0085] As a result, in this experiment, the tumor volumes of the SYJS001-ADC 0.5, 1, 2, 4 mg / kg (single administration) groups, IMAB362-ADC 0.5, 1, 2, 4 mg / kg (single administration) groups, and SYJS001-mAb 4 mg / kg (single administration) group were shown to be 59.1%, 27.3%, 1.8%, 0%, 68.2%, 30%, 1.8%, 0%, and 83.6% of the solvent control group, respectively. Compared with the solvent control group, each group except the SYJS001-mAb 4 mg / kg group could significantly inhibit tumor growth (p<0.001). The in vivo tumor inhibitory effect of SYJS001-ADC on NUGC4-CLDN18.2 was not significantly different from that of IMAB362-ADC at high doses, but under low-dose (effective dose) conditions, the in vivo tumor inhibitory effect of SYJS001-ADC was significantly superior to that of IMAB362-ADC (see Figure 9).

[0086] 1.5 ADC Stability Study This experiment aims to study the in vitro metabolic stability of SYJS001-ADC in plasma of different species (human, cynomolgus monkey, and Sprague-Dawley rat). The incubation concentration of SYJS001-ADC was 100 μg / mL, and it was incubated for 0 - 168 hours (7 days) under sterile conditions at 37°C. As a result of detecting the concentration of MMAE by LC-MS / MS method, the MMAE concentrations in 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma, and Sprague-Dawley rat plasma were as shown in Tables 2 - 5. With the increase of incubation time, the generation of free MMAE was observed in all substrates. After incubating SYJS001-ADC at 37°C for 168 hours (7 days), the dissociation rates in 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma, and Sprague-Dawley rat plasma were 0.672%, 0.327%, 0.209%, and 0.405% respectively in percentage. As is clear from the experiment, the dissociation rates of MMAE from SYJS001-ADC were all less than 1.0%, indicating that SYJS001-ADC is relatively stable in any of 0.5% BSA-PBS, human plasma, cynomolgus monkey plasma, and Sprague-Dawley rat plasma.

[0087]

Table 2

[0088]

Table 3

[0089]

Table 4

[0090]

Table 5

[0091] In addition, the inventors of the present invention also conducted an accelerated stability experiment. The specific steps are as follows. SYJS001-ADC was stored in a buffer solution (the components of which include 5% w / v L-histidine and 25% L-histidine hydrochloride) at 2°C - 8°C for different periods of time, and its stability was detected. The results are shown in the following table. IMAB362-ADC (IMAB362-vcMMAE) reached 0.3% of free MMAE in 28 days (see Table 7 of CN107667118A), which is much higher than 0.000026% of the drug SYJS001-ADC of the present application in three months, indicating that the stability of SYJS001-ADC is much better than that of IMAB362-ADC.

[0092]

Table 6

[0093] 1.6 Preparation of SYJS001-ADC drug composition The preparation steps of the drug composition (taking the formulation of 10 mg / ml SYJS001-ADC, 20 mmol / L histidine-histidine hydrochloride, 0.02% polysorbate 20, 6% sucrose, pH 5.5 as an example) are as follows.

[0094] (1) Affinity chromatography: The anti-CLDN18.2 antibody drug conjugate SYJS001-ADC is a conjugate of an antibody and a drug, contains one anti-CLDN18.2 antibody, and can be affinity-captured by Protein A. The anti-CLDN18.2 antibody drug conjugate SYJS001-ADC is captured using an affinity chromatography column (Mabselect sure chromatography medium), and other components LND1002 and mTgase directly flow through. After washing the chromatography column, it is eluted using a low pH elution buffer, and the eluate is collected in a sterile container.

[0095] (2) Liquid replacement by ultrafiltration: Prepare a histidine-histidine hydrochloride solution with a pH of 5.5 to a concentration equivalent to the formulation amount of the final product. Using a 30 kD ultrafiltration membrane pack, set the replacement ratio of liquid replacement by ultrafiltration to 100 times or more, control the pH value of the replacement liquid to match that of the final product, and perform liquid replacement so that the concentration of the anti-CLDN18.2 antibody drug conjugate SYJS001-ADC is 1.2 times or more the concentration of the final product, and replace the affinity chromatography solution with the histidine-histidine hydrochloride solution.

[0096] (3) Preparation of semi-finished product diluent: Calculate the preparation amount of the final product based on the formulation amount of the anti-CLDN18.2 antibody drug conjugate SYJS001-ADC and the drug solution concentration of the anti-CLDN18.2 antibody drug conjugate SYJS001-ADC after liquid replacement by ultrafiltration, calculate the required amount of each auxiliary material, and prepare a semi-finished product dilution solution. Add about 80% of water for injection to a blending container, weigh histidine, histidine hydrochloride, polysorbate 20, and sucrose in sequence and add them to the blending container, stir and mix. After each auxiliary material is completely dissolved, add water for injection to make up the volume to the preparation amount, stir and mix uniformly, and then filter for use.

[0097] (4) Manufacture of finished product: Add the prepared semi-finished product diluent to the preparation solution containing the anti-CLDN18.2 antibody drug conjugate SYJS001-ADC drug solution that has been liquid-replaced by ultrafiltration, make up the volume to the preparation amount of the semi-finished product with the diluent, stir and mix uniformly, sample to detect the protein content, pH value and microbial limit, perform sterile filtration, and then dispense into sterile injection bottles according to the product specifications, stopper and cap.

[0098] Example 2: Experiment on the effect of pH value selection on the stability of drug formulations The pH value is an important factor affecting the stability of biopharmaceutical liquid preparations. It has a regulatory effect on the charge distribution on the protein surface, affects the intermolecular and intramolecular forces of proteins, and influences the conformational stability, colloidal stability, and chemical stability of proteins. In this study, 10 mg / ml SYJS001-ADC samples with different pH values (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) were prepared using 20 mM citrate-phosphate as the buffer system, dispensed into 2-ml vial bottles, sealed with rubber stoppers, and stability studies were carried out under high-temperature (40 °C) conditions to evaluate the thermal stability (DSC), physical stability (SEC-HPLC), chemical stability (CEX-HPLC), and the content of free toxin. From the test results, it can be seen that the stability is excellent in the range of pH 5.5 - 6.5.

[0099] Detection method: DSC: The test sample was diluted with the same buffer until the protein concentration reached 1 mg / ml. 350 μl of each test sample and the corresponding buffer were taken and added to a 96-well plate at the corresponding positions. The 96-well plate was placed in the sample cell, and the temperature of the sample cell was set to 15 °C. Instrument parameter settings: starting temperature 20 °C, ending temperature 100 °C, heating rate 90 °C / min.

[0100] SEC-HPLC: In accordance with the general rule 0512 High Performance Liquid Chromatography in the fourth part of the Chinese Pharmacopoeia 2020 edition, analysis and detection were carried out using a column filled with a chromatography gel suitable for the separation of proteins with a molecular weight of 10 - 500 kD as the filler, and the ratios of the monomer peak, high molecular weight substance (HMWS) peak, and low molecular weight substance (HMWS) peak of the protein were calculated by the area normalization method.

[0101] CEX-HPLC: In accordance with the general rule 0512 High Performance Liquid Chromatography in the fourth part of the Chinese Pharmacopoeia 2020 edition, analysis and detection were carried out using a cation exchange column as the column, and the ratios of the acidic peak, main peak, and basic peak of the test sample were calculated by the area normalization method.

[0102] Free LND1002 content: In accordance with General Rule 0512, High Performance Liquid Chromatography, of the Fourth Part of the Chinese Pharmacopoeia 2020 Edition, it was precipitated with acetonitrile using a column packed with octadecylsilyl silica gel as the filler. After an ice bath, the supernatant was taken for analysis and detection, and the free LND1002 content in the test sample was calculated by the external standard method.

[0103] The detection results are as follows.

[0104]

Table 7

[0105]

Table 8

[0106]

Table 9

[0107]

Table 10

[0108] Example 3: Experiment on the effect of buffer solution on the stability of drug composition Buffer salts have an important impact on maintaining the pH value, physicochemical properties, and stability of the formulation. Buffer salts within the optimal pH range, such as citric acid-sodium citrate, succinic acid-sodium succinate, and histidine-histidine hydrochloride, are selected as buffer components, and the screening range includes but is not limited to 10 - 40 mmol / L. Samples with different buffer salts and different salt concentrations were prepared, with the ADC concentration being 10 mg / ml. They were dispensed into injection bottles, and stability studies were carried out under high-temperature (40°C) conditions. In the test, pH 6.0, 10 mg / ml SYJS001 samples were prepared with three buffer systems (citric acid, succinic acid, histidine) at different ion concentrations (10 mM, 20 mM, 40 mM), and the thermal stability, physical stability, chemical stability, and free toxin content were evaluated. The detection method was the same as in Example 2. As can be seen from the test results, the thermal stability in the three buffer systems is succinic acid > citric acid > histidine, and also, the lower the salt concentration, the better the thermal stability. There is no significant difference in physical stability and chemical stability. The free LND1002 content in both the succinic acid and histidine systems is lower than that in citric acid. It is preferable to use the histidine and succinic acid buffer systems as the basic buffer, and the concentration is preferably 20 mmol / L.

[0109] The specific detection results are as follows.

[0110]

Table 11

[0111]

Table 12

[0112]

Table 13

[0113]

Table 14

[0114] Example 4: Experiment on the Influence of Surfactants and Stabilizers on the Stability of Drug Compositions Based on the preferred pH value (6.0) and buffer salt (histidine-histidine hydrochloride), polysorbate 20, polysorbate 80, and sucrose, trehalose, mannitol, and sorbitol were added. The ADC concentration was set at 10 mg / ml, and the samples were dispensed into injection bottles. Stability studies were conducted under high-temperature (40 °C) conditions to evaluate thermal stability, physical stability, and chemical stability. The detection method was the same as in Example 2. As can be seen from the test results, by adding stabilizers, the thermal stability of the product can be improved, and the molecular size variants and charge variants of the samples in each group did not change significantly under the existing treatment conditions, indicating that the stability of this product is good. In addition to adding surfactants, it is preferable to use saccharides as stabilizers. The specific detection results are as follows.

[0115]

Table 15

[0116]

Table 16

[0117]

Table 17

[0118] Example 5: Experiment to Confirm Prescription Optimization Based on the results of the previous formulation tests, the interactions among various factors such as pH value, buffer salt, surfactant, and stabilizer were comprehensively considered. Here, the pH values were 5.5, 6.0, and 6.5; the buffer salts were succinic acid and histidine with a concentration of 20 mmol / L; the surfactants were polysorbate 20 and polysorbate 80 with a concentration of 0.02%; the stabilizers were sucrose and trehalose with a concentration of 6%. A total of 12 sets of formulations were designed for the test. The ADC concentration was set at 10 mg / ml. Samples of each set were dispensed into injection bottles, and stability studies were conducted under the conditions of high temperature (45°C) and light irradiation (4500 lx ± 500 lx) to evaluate the thermal stability, physical stability, chemical stability, and content of free toxins. The detection method was the same as that in Example 2. The test results were comprehensively analyzed, and the changes in Tm2, SEC monomer purity at the end points of light irradiation and high temperature tests, CEX main peak purity, and free toxin content of each set of formulations were used as the judgment conditions. As a result, it is shown that the pH value and the type of buffer salt have a relatively high impact on product quality, while the surfactant and stabilizer do not have a significant impact. However, when analyzing the overall formulation composition of each set, the degradation rate is not significant even under strong destruction conditions, indicating that this product has favorable stability in the above-mentioned formulation composition. According to the comprehensive analysis of the tests, since the impact of pH value and buffer salt on the product is relatively high, it is preferable to use the combination of 10 mg / ml SYJS001-ADC, 20 mmol / L histidine, 0.02% polysorbate 20, 6% sucrose, and pH 5.5 as the formulation for preparing clinical samples.

[0119]

Table 18

[0120]

Table 19

[0121]

Table 20

[0122]

Table 21

[0123]

Table 22

[0124] Example 6: Study on Prescription Stability 6.1 Study on High Temperature and Light Irradiation Stability One set of samples was prepared with a confirmed prescription of 10 mg / ml SYJS001-ADC, 20 mmol / L histidine, 0.02% polysorbate 20, 6% sucrose, and pH 5.5, and a preliminary stability study was conducted on it. The treatment conditions and detection items are shown in the following table.

[0125]

Table 23

[0126] As is clear from the above research results, the drug formulation of the present application does not undergo significant decomposition under high temperature and light irradiation conditions, and the quality of the prescription is stable and reliable.

[0127] 6.2 Study on Long-Term Stability Three batches of samples were prepared under GMP conditions with an optimized prescription composition of 10 mg / ml SYJS001-ADC, 20 mmol / L histidine-histidine hydrochloride, 0.02% polysorbate 20, 6% sucrose, and the remaining components being water for injection and pH 5.5. A stability study was conducted on the three batches of samples. The detection methods for each research index are all accurate and reliable methods verified by the methodology. The three batches of samples were simultaneously subjected to stability studies at -20°C ± 5°C and 5°C ± 3°C. Furthermore, the injection solution was subjected to a long-term stability study at -20°C ± 5°C and 5°C ± 3°C.

[0128] Detection method: SEC-HPLC: In accordance with the general chapter 0512 High Performance Liquid Chromatography in the fourth volume of the Chinese Pharmacopoeia 2020 edition, a column filled with a chromatographic gel suitable for separating proteins with molecular weights of 10 - 500 kD as the packing material (for example, XBridge® BEH 200 Å, 7.8×300 mm, 200 Å, 3.5 μm or other suitable columns) was used. A mobile phase consisting of 100 mmol / L phosphate buffer, 100 mmol / L NaCl, 10% isopropanol, pH 6.7 ± 0.1 was used, with a flow rate of 0.8 ml / min and a detection wavelength of 280 nm. 30 μg ± 3 μg of the test sample solution was taken, injected into the liquid chromatograph, and the ratios of the monomer peak, high molecular weight substance (HMWS) peak, and low molecular weight substance (HMWS) peak of the protein were calculated by the area normalization method.

[0129] CEX-HPLC: In accordance with the general chapter 0512 High Performance Liquid Chromatography in the fourth volume of the Chinese Pharmacopoeia 2020 edition, an ion exchange column (for example, MAbPac TM SCX-10, 4×250 mm or other suitable columns) was used as the column. 20 mmol / L CAPSO, 20 mmol / L NaCl, pH 8.0 was used as mobile phase A, 20 mmol / L CAPSO, 300 mmol / L NaCl, pH 10.0 was used as mobile phase B, with a flow rate of 1.0 ml / min and a detection wavelength of 280 nm. 40 μg ± 4 μg of the test sample solution was taken, injected into the liquid chromatograph for gradient elution, and the ratios of the acidic peak, main peak, and basic peak of the test sample were calculated by the area normalization method.

[0130] R-CE-SDS: Detected in accordance with the method for measuring molecular size variants of monoclonal antibodies in the general chapter 3127 of the fourth volume of the Chinese Pharmacopoeia 2020 edition, and the ratios of the heavy chain and light chain peaks and the ratio of the non-glycosylated heavy chain peak were calculated by the area normalization method.

[0131] NR-CE-SDS: Detected in accordance with the method for measuring molecular size variants of monoclonal antibodies in the general chapter 3127 of the fourth volume of the Chinese Pharmacopoeia 2020 edition, and the ratios of the monomer peak and the low molecular weight substance (2HC1LC, 1HC1LC) peak were calculated by the area normalization method.

[0132] DAR distribution: In accordance with General Chapter 0512 High Performance Liquid Chromatography of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), a column filled with tetraalkyl-bonded silica gel as the filler (for example, PLRP-S 1000A, 2.1×50 mm, particle size 5 μm or other suitable columns) was used. An aqueous solution of 0.1% (v / v) TFA was used as mobile phase A, an acetonitrile solution of 0.1% (v / v) TFA was used as mobile phase B, the column temperature was set at 80 °C, the flow rate was 0.25 ml / min, and the detection wavelength was 280 nm. 10 μL (10 μg ± 1 μg) of the test solution was taken and injected into the liquid chromatograph for gradient elution, and the DAR value and the DAR2 content were calculated.

[0133] Amount of uncomplexed antibody: Similar to the detection method of DAR distribution, it was analyzed by the external standard method using a spike control, and the amount of uncomplexed antibody was calculated by the external standard method.

[0134] Content of free LND1002: In accordance with General Chapter 0512 High Performance Liquid Chromatography of the Fourth Part of the Chinese Pharmacopoeia (2020 Edition), a column filled with octadecyl-bonded silica gel as the filler (for example, Agilent Poshocell 120 SB-C18, 4.6×150 mm, particle size 2.7 μm or other suitable columns) was used. An aqueous solution containing 0.1% TFA was used as mobile phase A, an acetonitrile solution containing 0.1% TFA was used as mobile phase B, the column temperature was set at 30 °C, the flow rate was 0.5 ml / min, and the detection wavelength was 250 nm. 100 μL (10 mg / mL) of the test sample was taken, precipitated with acetonitrile at a ratio of 1:3, placed at -20 °C, ice-bathed for 60 min, then centrifuged, and the supernatant was taken for loading. A certain amount of LND1002 (loading amount 65 ng) was used as the external standard control for gradient elution, and the content of free LND1002 in the test sample was calculated by the external standard method.

[0135] Content of free LND002 (%) = LND1002 detection concentration / ADC concentration × 100% Free MMAE content: In accordance with the general rules 0512 High Performance Liquid Chromatography and 0431 Mass Spectrometry in the fourth part of the Chinese Pharmacopoeia 2020 Edition, using a column packed with octadecyl-bonded silica gel (Waters ACQUITY UPLC (registered trademark) BEH - C18, 2.1×50 mm, particle size 1.7 μm or other suitable columns), an aqueous solution containing 0.1% TFA was used as mobile phase A, an acetonitrile solution containing 0.1% TFA was used as mobile phase B, the column temperature was set at 40 °C, the flow rate was 0.4 ml / min, and the detection wavelength was 250 nm. 50 μL (10 mg / mL) of the test sample was taken, the internal standard working solution was added, and precipitation treatment was carried out at a ratio of 1:3 with acetonitrile, placed at -20 °C, ice-bathed for 60 min, then centrifuged to take the supernatant for loading. Different concentrations of MMAE were used as reference substances for loading. After gradient elution of the sample by liquid chromatography, it was introduced into the mass spectrometer. After setting parameters such as the mass spectrometry capillary and cone voltage, ion source temperature, collision energy, and nebulization flow rate, analysis was carried out, and the free MMAE content in the test sample was calculated based on the standard curve.

[0136] Free MAE content (%) = MMAE detection concentration / ADC concentration × 100% Antigen-binding activity: Measured in accordance with the enzyme-linked immunosorbent assay (ELISA).

[0137] Biological activity: Measured in accordance with the cell growth inhibition method.

[0138] Detection results:

[0139]

Table 24

[0140]

Table 25

[0141]

Table 26

[0142]

Table 27

[0143]

Table 28

[0144]

Table 29

[0145] As is clear from the above research results, in the relatively low-temperature freezing state, up to 30 months, the physicochemical properties of the antibody part do not change significantly to match the stability trend of the antibody liquid preparation, neither the DAR value nor the DAR2 distribution of the ADC part changes significantly, indicating that the quality of the complex during storage is uniform. Although the release of the free drug is observed, the amount of release is very small, and the content of free LND1002 is less than 0.004% in all cases, and the content of free MMAE is less than 0.0015% in all cases, enabling long-term storage. The antigen-binding activity and biological activity do not change depending on the storage conditions. Also, according to the data of different batches, the consistency between batches is good, the long-term stability is good, and overall, it shows an ideal technical effect.

[0146]

Table 30(1)

Table 30(2)

Claims

1. A drug composition comprising an anti-CLDN18.2 antibody-drug conjugate, a buffer, a stabilizer, and a surfactant, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprises HCDR1-HCDR3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and / or the light chain comprises LCDR1-LCDR3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and the structure of the drug & linker moiety (L&D) in the antibody-drug conjugate is as shown below. 【Chemical 1】

2. The drug composition according to claim 1, comprising one or more of the following features. (a) The surfactant is polysorbate 20 or polysorbate 80, preferably polysorbate 20. (b) The stabilizer is sucrose or trehalose, preferably sucrose. (c) The buffer is histidine-histidine hydrochloride, succinic acid-sodium succinate, or citric acid-sodium citrate, preferably histidine-histidine hydrochloride.

3. 5-30 mM of histidine-histidine hydrochloride, 0.02-0.08 wt% of polysorbate 20 or polysorbate 80, 6-8 wt% of sucrose, and 5-50 mg / ml of the antibody-drug conjugate, The drug composition according to any one of claims 1-2, having a pH of 5.0-6.

5.

4. 10-30 mM of histidine-histidine hydrochloride, 0.02-0.04 wt% of polysorbate 20 or polysorbate 80, 6-7 wt% of sucrose, and 10-20 mg / ml of the antibody-drug conjugate, The drug composition according to any one of claims 1-2, having a pH of 5.0-6.

0.

5. 20 mM of histidine-histidine hydrochloride, 0.02 wt% of polysorbate 20, 6 wt% of sucrose, and 10 mg / ml of the antibody-drug conjugate, The drug composition according to any one of claims 1-2, having a pH of 5.

5.

6. The drug composition according to any one of claims 1-5, further comprising water.

7. The drug composition according to any one of claims 1-6, which is a liquid preparation, a freeze-dried preparation, or a powder injection preparation, preferably a liquid preparation.

8. The drug composition according to any one of claims 1 to 7, wherein the heavy chain of the antibody contains the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 7, and / or the light chain of the antibody contains the light chain variable region of the amino acid sequence shown in SEQ ID NO:

8.

9. The drug composition according to any one of claims 1 to 8, which is used for the treatment of cancer, preferably CLDN18.2-positive cancer.

10. The drug composition according to claim 9, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, endometrial cancer, uterine cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, multiple myeloma, neuroepithelial tissue tumor, schwannoma, head and neck cancer, skin cancer, laryngeal cancer, gallbladder cancer, cholangiocarcinoma, mesothelioma and sarcoma.

11. The drug composition according to claim 9, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer and uterine carcinosarcoma.

12. The drug composition according to claim 9, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma and Paget's disease.

13. The drug composition according to claim 9, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer and non-small cell lung cancer.

14. The drug composition according to any one of claims 9 to 13, which is used for administration in combination with other therapies, such as surgery, radiotherapy or hormonal therapy.

15. The drug composition according to any one of claims 9 to 13, which is used for administration in combination with other cancer therapeutics.

16. The drug composition according to claim 15, wherein the other cancer therapeutic is selected from 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT 11), docetaxel, pemetrexed, sorafenib, vinblastine, vinorelbine, everolimus, tanespimycin, bevacizumab, oxaliplatin and lapatinib.

17. Use of the pharmaceutical composition according to any one of claims 1 to 8 in the preparation of a drug for treating cancer, wherein the cancer is preferably a CLDN18.2 positive cancer.

18. The use according to claim 17, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, endometrial cancer, uterine cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, multiple myeloma, neuroepithelial tissue tumor, schwannoma, head and neck cancer, skin cancer, laryngeal cancer, gallbladder cancer, cholangiocarcinoma, mesothelioma and sarcoma.

19. The use according to claim 17, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer and uterine carcinosarcoma.

20. The use according to claim 17, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma and Paget's disease.

21. The use according to claim 17, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer and non-small cell lung cancer.

22. The use according to any one of claims 17 to 21, wherein the pharmaceutical composition is used for administration in combination with other therapies, such as surgery, radiotherapy or hormonal therapy.

23. The use according to any one of claims 17 to 21, wherein the pharmaceutical composition is used for administration in combination with other cancer therapeutic agents.

24. The use according to claim 23, wherein the other cancer therapeutic agent is selected from 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT 11), docetaxel, pemetrexed, sorafenib, vinblastine, vinorelbine, everolimus, tanespimycin, bevacizumab, oxaliplatin and lapatinib.

24. A method for treating cancer, comprising administering to a cancer-afflicted individual in need thereof an effective amount of the pharmaceutical composition according to any one of claims 1 to 8, wherein the cancer is preferably a CLDN18.2 positive cancer.

25. The method according to claim 24, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, endometrial cancer, uterine cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, multiple myeloma, neuroepithelial tissue tumor, schwannoma, head and neck cancer, skin cancer, laryngeal cancer, gallbladder cancer, cholangiocarcinoma, mesothelioma and sarcoma.

26. The method according to claim 24, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer and uterine carcinosarcoma.

27. The method according to claim 24, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer, non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, cholangiocarcinoma and Paget's disease.

28. The method according to claim 24, wherein the cancer is selected from breast cancer, gastric cancer, colorectal cancer and non-small cell lung cancer.

29. The method according to any one of claims 24 to 28, further comprising performing another therapy on the individual, such as surgery, radiation therapy or hormone therapy.

30. The method according to any one of claims 24 to 28, further comprising administering another cancer therapeutic agent to the individual.

31. The method according to claim 30, wherein the other cancer therapeutic agent is selected from 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT 11), docetaxel, pemetrexed, sorafenib, vinblastine, vinorelbine, everolimus, tanespimycin, bevacizumab, oxaliplatin and lapatinib.

32. A method for preparing the pharmaceutical composition according to any one of claims 1 to 8, comprising the steps of preparing the anti-CLDN18.2 antibody-drug conjugate, liquid displacement of the anti-CLDN18.2 antibody-drug conjugate into a liquid system containing other components of the pharmaceutical composition, and sterile filtration and aseptic filling.

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