Formulations containing antibody-drug conjugates targeting CLAUDIN18.2, methods for preparing the same, and use thereof.

Abstract

The present invention relates to a pharmaceutical formulation comprising an antibody-drug conjugate targeting Claudin 18.2, a method for preparing the pharmaceutical formulation, and the use of the pharmaceutical formulation for the treatment and / or prevention.

Description

[Technical Field]

[0001] The present invention relates to the field of pharmaceutical formulations of antibody-drug conjugates (ADCs) targeting Claudin18.2 (CLDN18.2). More specifically, the present invention relates to pharmaceutical formulations comprising antibody-drug conjugates targeting Claudin18.2, particularly lyophilized formulations, liquid formulations and reconstituted liquid formulations, methods for preparing pharmaceutical formulations, and the use of pharmaceutical formulations for the treatment and / or prevention. [Background technology]

[0002] Claudins are a family of proteins that are essential components of tight junctions between cells. They construct intercellular barriers that control the flow of molecules between cells, and these barriers can regulate the flow of molecules between cells. Claudins family proteins have tetraspanin domains, and their N-terminus and C-terminus are located in the cytoplasm. Different Claudins proteins are expressed in different tissues, and changes in their function are associated with the development of cancer in various tissues. For example, Claudin-1 is expressed in colon cancer and has a prognostic value, Claudin-18 is highly expressed in gastric and pancreatic cancer, and Claudin-10 is highly expressed in hepatocellular carcinoma. As cell membrane surface proteins, Claudins are useful targets for various therapeutic strategies.

[0003] Claudin-18 isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. Its expression in normal tissues is strictly limited to differentiated epithelial cells of the gastric mucosa, but it was not observed in gastric stem cell regions. CLDN18.2 is expressed in a significant portion of primary gastric cancers, and its expression levels are maintained in gastric metastatic cancer tissue. In addition to gastric cancer, CLDN18.2 expression is also observed in pancreatic cancer. CLDN18.2 is an ideal target molecule for the treatment of these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017)). https: / / doi.org / 10.1186 / s13045-017-0473-4 ).

[0004] Regarding gastric cancer, in 2014, China saw approximately 410,000 new cases and 290,000 deaths, accounting for nearly half of the total number of new cases and deaths worldwide, and the trend continues to increase. However, there are still many unmet needs in clinical oncology treatment, making the development of drugs targeting Claudin 18.2 extremely important.

[0005] Despite the clinical success achieved with therapeutic antibodies, naked MAbs targeting cell surface tumor antigens alone are rarely sufficient. To enhance the low activity of MAbs, new strategies focus on binding them to toxic molecules. Plant and bacterial toxins, as well as small molecules of chemotherapy, can be good candidates because they are highly potent and active even in very small amounts.

[0006] Technological advancements over the past few years have led to a recent increase in development activities by pharmaceutical companies in the field of antibody-drug conjugates (ADCs) for cancer treatment, addressing the initial problems associated with ADCs regarding immunogenicity, affinity, specificity, undesirable toxicity, yield, and half-life.

[0007] Despite some progress, additional treatment strategies for the treatment of tumors and the components used in such treatment strategies are still needed, especially anti-Claudin18.2 antibodies with high affinity, excellent specificity, and / or low risk of immunogenicity, and ADC molecules with higher activity, low toxicity, long half-life, excellent specificity or affinity, and / or excellent half-life or drug discovery potential.

[0008] There is a need for novel pharmaceutical formulations containing antibody-drug conjugates targeting Claudin18.2 that are sufficiently stable and suitable for administration. Pharmaceutical formulations, especially lyophilized formulations, have good stability, maintain a stable state during storage and transportation, and lyophilized formulations can be easily administered after reconstitution.

Summary of the Invention

[0009] The present invention provides a pharmaceutical formulation comprising an antibody-drug conjugate (ADC) targeting Claudin18.2.

[0010] In a first aspect, the present invention provides a pharmaceutical formulation comprising (i) an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof, (ii) a buffer, (iii) a stabilizer, and / or (iv) a surfactant, and the pH of the formulation is from about 5.5 to 7.5, such as from about 6.0 to 6.5, such as 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 or 7.2, preferably about 6.5.

[0011] In some embodiments, the Claudin18.2 antibody-drug conjugate is

Chemical formula

Chemical formula

[0012] Depending on the context, when the variable after the square brackets in the ADC structural formula is specified as the average DAR (e.g., q in the above structural formula), it should be understood that it represents the ratio of the drug moiety (e.g., exatecan) to Ab measured by the conventional measurement method, as described in the definition section of this specification. The average DAR can be represented as a decimal. For example, when expressing "q is from 3 to 5" or "q is a number from 3 to 5" for the average DAR, it means that q is any number including integers and decimals from 3 to 5.

[0013] In some embodiments, the Claudin18.2 antibody-drug conjugate is

Chemical formula

Chemical formula

[0014] In some embodiments, the antibody or antigen-binding fragment targeting Claudin18.2 includes HCDR1, HCDR2, and HCDR3, each consisting of the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences described in SEQ ID NOs: 6, 7, and 8, respectively. In some embodiments, an antibody targeting Claudin18.2 or its antigen-binding fragment includes a heavy chain variable region and / or a light chain variable region, the heavy chain variable region is (i) containing or consisting of the amino acid sequence described in Sequence ID No. 4, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in Sequence ID No. 4, or comprising such an amino acid sequence, (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No. 4, wherein preferably the amino acid modifications do not occur in the CDR and / or The light chain variable region is, (i) containing or consisting of the amino acid sequence described in Sequence ID No. 9, or (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in Sequence ID No. 9, or comprising such an amino acid sequence, (iii) The amino acid sequence comprises or consists of one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No. 9, and preferably the amino acid modifications do not occur in the CDR.

[0015] In some embodiments, an antibody targeting Claudin18.2 or its antigen-binding fragment comprises a heavy chain and a light chain. The heavy chain is, (i) containing or consisting of the amino acid sequence described in SEQ ID NO: 11, or (ii) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acids described in SEQ ID NO: 11, or comprising such an amino acid sequence, (iii) an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No. 11, or comprising such an amino acid sequence and / or Light chains are (i) containing or consisting of the amino acid sequence described in Sequence ID No. 12, or (ii) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in Sequence ID No. 12, or comprising such an amino acid sequence, (iii) an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No. 12, or comprising such an amino acid sequence.

[0016] In some embodiments, the buffer is selected from one or more of histidine, citrate, succinic acid, glutamic acid, phosphoric acid, acetic acid, or salts thereof, and preferably the buffer is a combination of histidine and histidine hydrochloride, or histidine.

[0017] In some embodiments, the stabilizer is selected from one or more carbohydrates and polyols, preferably the carbohydrate is selected from sucrose, trehalose, glucose, lactose, maltose, cyclodextrin, maltodextrin, and dextran, and the polyol is selected from mannitol, sorbitol, and xylitol, more preferably the stabilizer is selected from sucrose, trehalose, mannitol, or a combination thereof.

[0018] In some embodiments, the surfactant is selected from alkyl poly(ethylene oxide), polysorbate, pluronic, or a combination thereof, preferably from polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40.

[0019] In some embodiments, the buffer is a combination of histidine and histidine hydrochloride, or histidine alone. The stabilizers are sucrose, trehalose, or a combination of sucrose and mannitol, and / or The surfactant is polysorbate 80.

[0020] In some embodiments, the pharmaceutical formulation comprises an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof in a concentration of approximately 1 to 200 mg / ml, preferably 15 to 60 mg / ml, and more preferably approximately 20 to 40 mg / ml.

[0021] In some embodiments, the pharmaceutical formulation further comprises about 1 to 100 mM, preferably about 5 to 50 mM, and more preferably about 10 to 30 mM of buffer solution.

[0022] In some embodiments, the pharmaceutical formulation further comprises a stabilizer in an amount of about 10 to 200 mg / ml, preferably about 50 to 100 mg / ml, and more preferably about 60 to 90 mg / ml.

[0023] In some embodiments, the pharmaceutical formulation further comprises a surfactant in an amount of about 0.01 to 5 mg / ml, preferably about 0.05 to 1 mg / ml, and more preferably about 0.1 to 0.3 mg / ml.

[0024] In some embodiments, the pharmaceutical formulation is (i) Approximately 20-40 mg / ml of an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof. (ii) Approximately 20 mM buffer solution, (iii) A stabilizer of approximately 60-90 mg / ml, and (iv) Contains approximately 0.1 to 0.3 mg / ml of surfactant.

[0025] In some embodiments, the pharmaceutical formulation is (i) Approximately 20-40 mg / ml of an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof. (ii) A buffer solution of approximately 20 mM, which is a combination of histidine and histidine hydrochloride, or histidine, (iii) A stabilizer of approximately 60-90 mg / ml, which is sucrose, trehalose, or a combination of sucrose and mannitol, and (iv) Contains approximately 0.1-0.3 mg / ml of polysorbate 80, The pH of the liquid is approximately 6.0 to 6.5, preferably approximately 6.5.

[0026] In some embodiments, the pharmaceutical formulation is (i) Approximately 20 mg / ml of an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof. (ii) A buffer solution of approximately 20 mM, which is a combination of histidine and histidine hydrochloride, or histidine, (iii) A stabilizer of approximately 8% (w / v), which is sucrose, trehalose, or a combination of sucrose and mannitol, and (iv) Contains approximately 0.02% (w / v) of polysorbate 80, The pH of the formulation is approximately 6.5.

[0027] In a second aspect, the present invention provides a lyophilized pharmaceutical formulation comprising an antibody-drug conjugate, which, after reconstitution, can form or provide the pharmaceutical formulation described in the first aspect.

[0028] In some embodiments, the lyophilized pharmaceutical formulation is prepared by lyophilizing the pharmaceutical formulation described in the first embodiment above.

[0029] In a third aspect, the present invention provides a lyophilized pharmaceutical formulation containing an antibody-drug conjugate, which is (i) an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof in an amount of approximately 1% to 60% (w / w), preferably approximately 15% to 40% (w / w), (ii) A buffer solution of approximately 0.5% to 10% (w / w), preferably approximately 2% to 5% (w / w), (iii) a stabilizer in an amount of about 20% to 90% (w / w), preferably about 50% to 85% (w / w), and / or (iv) containing approximately 0.05% to 2% (w / w), preferably approximately 0.1% to 0.5% (w / w), a surfactant, The antibody-drug conjugate, its pharmaceutically acceptable salt or solvate, buffer, stabilizer and / or surfactant are as defined in the first embodiment above.

[0030] In a fourth aspect, the present invention provides a liquid pharmaceutical formulation comprising an antibody-drug conjugate, which is obtained by reconstituting the above-mentioned lyophilized pharmaceutical formulation with a solvent, wherein the pH is about 5.5 to 7.5, preferably about 6.0 to 6.5, and more preferably about 6.5.

[0031] In some embodiments, the liquid pharmaceutical formulation is used for injection or infusion, such as intravenous injection, subcutaneous injection, or intramuscular injection.

[0032] In a fifth aspect, the present invention provides a delivery device comprising a pharmaceutical formulation, a lyophilized pharmaceutical formulation, or a liquid pharmaceutical formulation of the present invention.

[0033] In a sixth aspect, the present invention provides a pre-filled syringe for intravenous, intramuscular, or subcutaneous injection, comprising a pharmaceutical formulation of the present invention, a lyophilized pharmaceutical formulation, or a liquid pharmaceutical formulation.

[0034] In a seventh aspect, the present invention provides the use of the pharmaceutical formulation, lyophilized pharmaceutical formulation and liquid pharmaceutical formulation of the present invention in the manufacture of pharmaceuticals, preferably the pharmaceutical formulation, lyophilized pharmaceutical formulation and liquid pharmaceutical formulation being used to prevent or treat tumors.

[0035] More preferably, the tumor is cancer, such as epithelial carcinoma or gastrointestinal carcinoma, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.

[0036] In an eighth aspect, the present invention provides a pharmaceutical formulation, a lyophilized pharmaceutical formulation, or a liquid pharmaceutical formulation, as well as a pharmaceutical combination comprising one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

[0037] In a ninth aspect, the present invention provides a kit of parts comprising a pharmaceutical formulation, a lyophilized pharmaceutical formulation, or a liquid pharmaceutical formulation, or a pharmaceutical combination of the present invention, preferably the kit being in the form of a drug administration unit.

[0038] The antibody-drug conjugate formulations of the present invention that target Claudin18.2 have the following advantages and / or benefits: (1) The formulation of the present invention showed excellent stability against various factors affecting stability, such as temperature, vibration, freeze-thaw cycles, and light. Even after long-term storage or accelerated testing, no significant changes were observed in the appearance of the formulation, protein concentration, insoluble particles, DAR, etc., and good antibody activity was maintained, making it suitable for long-term storage or transport. (2) The lyophilized formulation of the present invention is rapidly reconstituted. The physicochemical properties and activity of the reconstituted antibody-drug conjugate remain largely unchanged, making it beneficial for administration to patients. (3) The preparation process for the formulation of the present invention is simple and highly reproducible.

[0039] Furthermore, the complex in the formulation of the present invention has the following advantages: (1) It binds to target cells expressing human CLDN18.2 and has high affinity, (2) The ADC of the present invention can enter the cell via endocytosis and kill the target cell, and in some embodiments, the ADC of the present invention has high endocytosis efficiency. (3) It has a significant bystander killing effect, (4) It has a high antitumor effect, (5) It has low toxicity, (6) It has good stability, (7) Has good potential for drug discovery. [Brief explanation of the drawing]

[0040] [Figure 1] This demonstrates the cell-binding effect of the IEX019 molecule. [Figure 2] This demonstrates endocytosis of the IEX019 molecule in DANG-hCLDN18.2 cells. [Figure 3A] This study demonstrates the killing effect of the IEX019 molecule in cell lines with low hCLDN18.2 expression. [Figure 3B] hCLDN18.2 expression indicates a killing effect of the IEX019 molecule in cell lines with moderate levels of expression. [Figure 3C] This study demonstrates the killing effect of the IEX019 molecule in cell lines with high hCLDN18.2 expression. [Figure 4] The IEX019 molecule demonstrates bystander-killing effects. [Figure 5] The tumor-suppressing effect (A) and body weight change (B) of the IEX019 molecule in mice are shown. [Figure 6]The tumor-suppressing effect (A) and body weight change (B) of the IEX019 molecule in mice are shown. [Figure 7] The tumor-suppressing effect (A) and body weight change (B) of the IEX019 molecule in mice are shown. [Figure 8] The DSC profile of the ADC pH buffer screening is shown. [Modes for carrying out the invention]

[0041] I. Definition Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and these may change. It should also be understood that the terms used herein are intended to describe specific embodiments only and do not limit the scope of the present invention, and the scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.

[0042] To illustrate this specification, the following definitions are used, and where appropriate, terms used in the singular form may also have a plural form, and vice versa. It should be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit them.

[0043] When used in conjunction with a number, the term "approximately" is intended to encompass a range of numbers from a lower limit of 5% below the specified number to an upper limit of 5% above the specified number.

[0044] As used herein, the term "and / or" refers to one or more of the options.

[0045] As used herein, the terms “comprise” or “include” mean that the elements, integers, or steps described are included, but not that other elements, integers, or steps are excluded. As used herein, the terms “comprise” or “include” also include situations that consist entirely of the elements, integers, or steps described, unless otherwise specified. For example, when referring to an antibody variable region of a particular sequence as “comprise,” it is intended to also include an antibody variable region consisting of that particular sequence.

[0046] As used herein, the terms “CLAUDIN” or “CLDN” refer to the most important skeletal proteins that determine the structure of intercellular tight junctions, are involved in adhesion junctions, and play a crucial role in tumor cell metastasis and invasion. Claudin proteins are widely present in mammalian epithelial and endothelial cells, primarily on the lateral surfaces of epithelial cells and on the cell membranes of basal cells. Different Claudin proteins are expressed specifically in different tissues, and the Claudin18 (CLDN18) gene is located at 3q22.3, has a molecular weight of 24 kDa, contains 261 amino acid residues, and is a member of the Claudins superfamily. Its protein structure includes two extracellular loops and four transmembrane regions. The two subtypes of the human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1) and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2), respectively. The primary structural sequences of the two proteins differ only in a few amino acid residues from the N-terminal signal peptide to the extracellular loop 1 (loop) structure. In particular, in extracellular loop 1, CLDN18.1 and CLDN18.2 differ by only eight amino acids. The intergeneric and interspecies sequence homology between the two subtypes of the CLDN18 protein is also very high. The extracellular loop 1 sequence of CLDN18.2 is completely identical in different species such as humans, mice, and rhesus monkeys, and the homology of human and mouse CLDN18.2 proteins reaches 84%, indicating that the sequence of the CLDN18.2 protein is extremely conserved (O. Tureci et al., Gene, 481:83-92, 2011). CLDN18.2 or any of its variants and isoforms can be isolated from cells or tissues that express it naturally, or recombinantly generated using techniques well known in the art and / or techniques described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0047] As used herein, the terms “anti-CLDN18.2 antibody,” “anti-CLDN18.2,” “CLDN18.2 antibody,” “antibody that binds to CLDN18.2,” “antibody that specifically binds to CLDN18.2,” “antibody that targets CLDN18.2,” or “CLDN18.2 targeting antibody” refer to antibodies that can bind to (human) CLDN18.2 with sufficient affinity to be used as therapeutic agents targeting (human) CLDN18.2. In one embodiment, the (human) CLDN18.2 antibody binds to (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, the (human) CLDN18.2 antibody does not bind to CLDN18.1. In one embodiment, the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1. In some embodiments, binding is determined by, for example, radioimmunoassay (RIA), biolayer interferometry (BLI), MSD assay, surface plasmon resonance (SPR), or flow cytometry. Intracellular CLDN18.2 expression can be determined by several methods, including anti-CLDN18.2 antibodies. For example, the binding strength between "highly expressing CLDN18.2" cells and anti-CLDN18.2 antibodies (determined, e.g., by FACS) may be 500, 600, 700, 800, 900 times or more, or preferably 1000 times or more, e.g., 1100 times or more, 1200 times or more, 1300 times or more, 1400 times or more, or more, than the binding strength between anti-CLDN18.2 antibodies and cells without CLDN18.2 expression. For example, the binding strength (determined, for example, by FACS) between cells with "moderate CLDN18.2 expression" and an anti-CLDN18.2 antibody is 5 to 500 times greater than the binding strength between the anti-CLDN18.2 antibody and cells without CLDN18.2 expression, for example, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more, but it may be less than 500 times.

[0048] The terms "whole antibody," "intact antibody," or "full-length antibody" are used interchangeably herein and refer to antibody molecules that have the structure of a natural immunoglobulin molecule. In the case of conventional four-chain IgG antibodies, a full-length antibody contains two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy-chain antibodies, only heavy chains are present and light chains are absent, and a full-length antibody contains two heavy chains (H) interconnected by disulfide bonds.

[0049] In conventional quadruple-chain IgG antibodies, the full-length antibody heavy chain generally consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, the heavy chain constant region containing at least three domains CH1, CH2, and CH3. The full-length antibody light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region, the light chain constant region consisting of one domain CL. Each heavy chain variable region VH or each light chain variable region 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, and FR4.

[0050] The term "antibody fragment" includes a portion of the entire antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment.

[0051] An "antigen-binding fragment" is a molecule distinct from the entire antibody, containing a portion of the entire antibody, and refers to a molecule that binds to the antigen to which the entire antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibodies), linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies (e.g., VHH), bivalent or bispecific antibodies or their fragments, and Camelidae antibodies.

[0052] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies, the activation of specific immune cells, or both. Technologists understand that virtually any macromolecule, including almost any protein or peptide, can be used as an antigen. Furthermore, antigens can also be generated from recombinant DNA or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0053] A "complementarity-determining region," "CDR region," or "CDR" is a region within the antibody variable domain that is highly sequence-variable, forms a structurally defined loop ("hypervariable loop"), and / or contains an antigen contact residue ("antigen contact site"). CDRs are primarily responsible for binding to the antigen epitope. Heavy chain and light chain CDRs are generally called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. CDRs located in the heavy chain variable domain of an antibody are called HCDR1, HCDR2, and HCDR3, and CDRs located in the light chain variable domain of an antibody are called LCDR1, LCDR2, and LCDR3.In a given amino acid sequence of the light chain variable region or heavy chain variable region, the precise amino acid sequence boundary of each CDR is determined, for example, by Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, Journal of Molecular Biology, 273:927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loop; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USD Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and the International ImMunoGeneTics database (IMGT) (World Wide). This can be determined using any one or a combination of many well-known antibody CDR numbering systems, including the North CDR definition based on affinity propagation clustering using numerous crystal structures (North et al, “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)) available on the web at imgt.cines.fr / .

[0054] The following is the scope of CDR as defined by the kabat, AbM, Chothia, Contact, and IMGT protocols. [Table 1]

[0055] Furthermore, the CDR may be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0056] Unless otherwise specified, the terms “CDR” or “CDR sequence” as used herein encompass CDR sequences determined by any of the methods described above.

[0057] Unless otherwise specified, the residue positions of the antibody variable region (including heavy chain variable region residues and light chain variable region residues) in this invention are positions numbered according to the Kabat numbering system (Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0058] In one embodiment, the heavy chain variable region (CDR) of the antibody of the present invention is determined according to the following scheme.

[0059] VH CDR1 is determined according to the AbM scheme, and VH CDR2 and 3 are determined according to the Kabat scheme.

[0060] In one embodiment, the light chain variable region (CDR) of the antibody of the present invention is determined according to the Kabat scheme.

[0061] In one embodiment, the heavy chain variable region CDR of the antibody of the present invention is determined according to the following scheme: VH CDR1 is determined according to the AbM scheme, VH CDR2 and 3 are determined according to the Kabat scheme, and each CDR of the light chain variable region is determined according to the Kabat scheme.

[0062] It should be noted that the CDR boundaries of the variable region of an antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of an antibody defined by different assignment systems will differ. Therefore, when defining an antibody having a specific CDR sequence as defined in this invention, the range of antibodies also includes antibodies whose variable region sequence contains a specific CDR sequence, but which, due to the different scheme applied (e.g., different assignment system schemes or combinations thereof), claim a different CDR boundary than the specific CDR boundary defined in this invention.

[0063] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although the CDR differs from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. The smallest overlapping region can be determined using at least two of the Kabat, Chothia, AbM, Contact, and North methods, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit may be a subpart of the CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody structure and protein folding. Therefore, any variant of the CDR described herein is also considered in the present invention. For example, in CDR variants, the amino acid residues within the minimum binding unit remain unchanged, while other CDR residues, as defined by Kabat or Chothia, may be substituted with conserved amino acid residues.

[0064] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. This term includes both the Fc region of the natural sequence and mutant Fc regions. The “Fc domain” of a natural immunoglobulin includes two or three constant domains, namely the CH2 and CH3 regions, and optionally the CH4 region. For example, the immunoglobulin Fc domain of a natural antibody includes second and third constant domains (CH2 and CH3 regions) derived from two heavy chains of IgG, IgA, and IgD, or second, third, and fourth constant domains (CH2, CH3, and CH4 regions) derived from two heavy chains of IgM and IgE. Unless otherwise specified, the amino acid residue numbers of the Fc region or constant region are based on the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this invention, the “Fc region” does not include the heavy chain variable region VH and light chain variable region VL, as well as the heavy chain constant region CH1 and light chain constant region CL of immunoglobulins, but may in some cases include the N-terminal hinge region of the heavy chain constant region.

[0065] The term "IgG-type antibody" is used to define the IgG type to which the antibody's heavy chain constant region belongs. Different types of antibodies have different heavy chain constant regions. For example, an IgG4-type antibody refers to one whose heavy chain constant region is derived from IgG4, while an IgG1-type antibody refers to one whose heavy chain constant region is derived from IgG1.

[0066] As used herein, the terms “binding” or “specific binding” mean that the binding interaction to an antigen is selective and can be distinguished from undesirable or nonspecific interactions. The ability of an antigen-binding site to bind to a particular antigen can be determined by enzyme immunosorbent assay (ELISA) or conventional binding assays known in the art (e.g., radioimmunoassay (RIA), thin-layer biomembrane interference assay, MSD assay, surface plasmon resonance (SPR), etc.).

[0067] As used herein, “antibody-drug conjugate (ADC)” refers to a structure / compound typically obtained by conjugating an antibody and a drug via a linker.

[0068] As used herein, the general terms “carbohydrate” or “sugar” refer to monocarbohydrates, such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). As used herein, the term “carbohydrate derivative” refers to a derivative of a monocarbohydrate, i.e., a monocarbohydrate containing substituents and / or functional groups. Examples of carbohydrate derivatives include aminocarbohydrates and carbohydrate acids, such as glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Examples of carbohydrate derivatives also include compounds denoted herein as E(A)x, where E is a carbohydrate or carbohydrate derivative, and E contains x functional groups A.

[0069] The core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as GlcNAc bound to the antibody via a C1, preferably via an N-glycosidic bond to the amide nitrogen atom of the side chain of the asparagine amino acid of the antibody. The core-GlcNAc substituent may be present at the original glycosylation site of the antibody, but may also be introduced at other sites on the antibody. herein, the core-N-acetylglucosamine substituent is either a monocarbohydrate substituent or (if the above core-GlcNAc substituent is fucosylated) a dicarbohydrate core-(Fucα1~6)GlcNAc substituent, also known as GlcNAc(Fuc).

[0070] "Glycosylation modification" refers to the process of altering the glycan chain of an antibody through glycosylation engineering. Antibody glycosylation can be further modified for various purposes to obtain antibodies with new glycosylations. For example, glycosylation can be removed to eliminate FcγR affinity and complement binding / effector function, fucose and sialic acid groups can be reduced, and bifurcation N-acetylglucosamine, galactose, and mannose can be added to enhance Fc-mediated ADCC and CDC effects. Methods of glycosylation modification are known in the art and include, for example, increasing or decreasing the saccharide chain(s) on the antibody surface by altering the glycosylation site of the antibody, modifying the saccharide chain(s) by in vitro chemical or enzymatic methods, catalyzing antibody glycosylation by altering the glycosylation pathway of the expression system (e.g., consisting of glycosidase and glycosyltransferase), and altering antibody glycosylation by affecting cell culture conditions. In some embodiments, the "glycosylation modification" of the present invention is carried out by modifying the saccharide chain(s) in an in vitro enzymatic manner. Preferably, the glycosylation modification of the present invention is carried out by modifying the saccharide chain(s) with a glycosidase (e.g., endoglycosidase or glycosyltransferase).

[0071] The modified glycosylation antibody of the present invention refers to an antibody having a modified glycosylation pattern compared to an antibody having a naturally occurring glycosylation pattern. Preferably, the modified glycosylation antibody refers to an antibody obtained by modifying the saccharide chain(s) of an antibody expressed in an expression system (e.g., mammalian cells) by an in vitro enzymatic method (e.g., modifying the saccharide chain(s)(s) with a glycosidase (e.g., endoglycosidase or glycosyltransferase)). More preferably, the modified glycosylation antibody of the present invention refers to an antibody comprising a core-GlcNAc and a carbohydrate derivative E(A)x bound thereto, wherein the GlcNAc is bound to the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom of the side chain of the asparagine amino acid of the antibody. When the GlcNAc in the GlcNAc-E(A)x substituent is fucosylated, the fucose is usually bound to C6 of the -GlcNAc substituent via α-1,6. The fucosylated GlcNAc substituent is denoted as core-GlcNAc(Fuc). The fucosylated GlcNAc-E(A)x substituent is denoted as GlcNAc(Fuc)-E(A)x.

[0072] As used herein, the term “site-directed conjugation” refers to a conjugation in which a drug / active substance specifically binds to a particular site on an antibody via a linker.

[0073] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Typical examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0074] The term "aryl" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 20 carbon atoms, for example, 6 to 12 carbon atoms, in the ring portion. Preferably, the aryl is (C6~C 10 ) is aryl. Examples that are not limited to include phenyl, biphenyl, naphthyl or tetrahydronaphthyl, each of which may be optionally substituted with 1 to 4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, sulfidyl, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonylamino, heterocyclyl, etc., where R is independently hydrogen, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, etc.

[0075] The term "cycloalkyl" refers to a cyclic alkyl group, i.e., a monovalent saturated or unsaturated hydrocarbon group having a cyclic structure. Cycloalkyls include all saturated or partially saturated (containing one or two double bonds) hydrocarbon groups having a cyclic structure. A cycloalkyl group may contain three or more carbon atoms in the ring, for example, 3 to 18, 3 to 10, or 3 to 8 carbon atoms, and according to the present invention, typically 3 to 6 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0076] As used herein, the term “heteroaryl” refers to a 5-20 member (e.g., 5-14, 5-8, 5-6 member) monocyclic, bicyclic, or fused polycyclic ring system containing 1-8 heteroatoms selected from N, O, or S. Preferably, heteroaryls are 5-10 member ring systems. Typical heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4- or 5-imidazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 3-1,2,4-triazolyl or 5-1,2,4-triazolyl, 4-1,2,3-triazolyl or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyridazinyl, 2-pyridazinyl, and 2-, 4- or 5-pyrimidinyl.

[0077] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC complex of the present invention and is not biologically or otherwise undesirable. The ADC complex of the present invention may exist in the form of pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed with the ADC complex of the present invention and an organic or inorganic acid, and includes, but is not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. A pharmaceutically acceptable, non-toxic base addition salt represents a salt formed with the ADC complex of the present invention and an organic or inorganic base, and includes, but is not limited to, alkali metal salts, such as lithium, sodium, or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and salts of organic bases, such as ammonium salts formed with N-group-containing organic bases.

[0078] The term "solvate" refers to an associated compound formed by one or more solvent molecules and the ADC complex of the present invention. Solvents for forming solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, and dimethyl sulfoxide.

[0079] Unless otherwise specified by context, "pharmaceutically acceptable" and "pharmaceutically usable" are used interchangeably herein.

[0080] The term “drug-to-antibody ratio” or “DAR” refers to the ratio of the small molecule drug moiety (D) bound to the Ab moiety to the Ab moiety, as described herein. In some embodiments described herein, for example, the DAR may be 1 to 20, e.g., 2 to 18, 4 to 16, 5 to 12, 6 to 10, 2 to 8, 3 to 8, 2 to 6, 4 to 6, 6 to 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the small molecule drug moiety (D) bound to the Ab moiety (D) to the Ab moiety in the product, as described herein, and measured by a detection method (e.g., conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), and such a DAR is referred to in this context as the average DAR. In some embodiments, the composite of the present invention has an average DAR value of 1 to 20, for example 2 to 18, 4 to 16, 5 to 12, 6 to 10, 2 to 8, 3 to 8, 2 to 6, 4 to 6, 6 to 10, for example 1.0 to 8.0, 2.0 to 6.0, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4. The range is 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, with two of these values ​​as the endpoints.

[0081] As used herein, the term “therapeutic agent” encompasses any substance effective in preventing or treating tumors such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0082] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. In some contexts, it may also be called a “toxin.”

[0083] A "free agent" is an antitumor compound or cytotoxic agent in its free form, dissociated from the ADC molecule of the present invention. Depending on the context, a free agent may also be called a free toxin.

[0084] "Chemotherapy agents" include compounds useful for treating cancer or immune system disorders.

[0085] The term "small molecule drugs" refers to organic compounds with low molecular weight that can modulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetic and antibody mimetic. As therapeutic agents, small molecules are thought to have higher cell permeability, be less prone to degradation, and be less likely to induce an immune response compared to large molecules.

[0086] As used herein, the term “immunomodulator” refers to a natural or synthetic activator or agent that suppresses or modulates an immune response. An immune response may be a humoral or cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0087] The term "effective dose" refers to the amount or dosage of the antibody-drug conjugate or its pharmaceutically acceptable salt, antibody, fragment, composition, or combination of the present invention, which, after being administered to a patient or subject in a single or multiple dose, produces the expected effect in a patient or subject requiring treatment or prevention.

[0088] The "therapeutic dose" refers to the amount that effectively achieves the desired therapeutic outcome for the required duration and in the required dose. The therapeutic dose is also the amount in which the toxicity or adverse effects of the antibody-drug conjugate or its pharmaceutically acceptable salt, antibody, antibody fragment, composition, or combination outweigh the beneficial therapeutic effect. The "therapeutic dose" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, and more preferably by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% compared to an untreated subject.

[0089] The "prophylactic effective dose" refers to the amount that can effectively achieve the desired preventive outcome for the required period and in the required dosage. Generally, since prophylactic doses are administered before or in the early stages of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0090] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may not be exactly the same as parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity, sorted or selected from the initially transformed cells, are included herein.

[0091] As used herein, the term “label” refers to a compound or composition that is directly or indirectly bound or fused to a drug (such as a polynucleotide probe or antibody) to facilitate the detection of the bound or fused drug. The label itself may be detectable (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, may catalyze a chemical change in a detectable substrate compound or composition. This term is intended to cover both direct labeling of a probe or antibody by binding (physically connecting) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reacting with another directly labeled reagent.

[0092] "Individual" or "Subject" includes mammals. Mammals include, but are not limited to, livestock (cattle, sheep, cats, dogs, horses, etc.), primates (humans and non-human primates, e.g., monkeys), rabbits, and rodents (mice and rats, etc.). In some embodiments, the individual or subject is a human.

[0093] "Isolated" antibodies or other molecules (e.g., ADC molecules) are antibodies or molecules that have been separated from components of the natural environment or the environment in which they are expressed. In some embodiments, the antibody or ADC molecule is purified to a purity of 95% or greater than 99% by means of electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0094] The term "antitumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival rate.

[0095] In this specification, the terms “tumor” and “cancer” may be used interchangeably to include solid tumors and hematological malignancies.

[0096] The terms “cancer” and “cancerous” refer to or describe a physiological disorder of mammals characterized by uncontrolled cell proliferation. In some embodiments, cancers suitable for treatment with the antibody-drug conjugates of the present invention include gastric cancer, pancreatic cancer, or gastroesophageal junction cancer (including metastatic forms of these cancers).

[0097] The term “tumor” refers to the growth and proliferation of all tumor cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” as used herein are not mutually exclusive.

[0098] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, stabilizers, etc. See Handbook of Pharmaceutical Excipients, 8th Ed, RCRowe, PJSeskey and SCOwen, Pharmaceutical Press, London, Chicago.

[0099] The term "pharmaceutical composition" refers to a composition in which the active ingredients contained herein exist in a form that enables their effective biological activity, and which does not contain any additional ingredients that would be unacceptably toxic to the subject to which the composition is administered.

[0100] The term “pharmaceutical combination” refers to a non-fixed or fixed combination product, including but not limited to kits and pharmaceutical compositions. “Non-fixed combination” means that the active ingredients (e.g., (i) the ADC molecule in the pharmaceutical formulation of the present invention, and (ii) other therapeutic agents) are administered to the patient simultaneously or sequentially (without specific time limitations, or at the same or different time intervals) as separate entities, and such administration provides the patient with prophylactic or therapeutically effective levels of two or more active ingredients. In some embodiments, the ADC molecule and other therapeutic agents used in the pharmaceutical combination are administered at concentrations not exceeding those obtained when each is used alone. “Fixed combination” means that two or more active ingredients are administered to the patient simultaneously in the form of a single entity. In the treatment of a disease or disorder, it is preferable to select doses and / or time intervals of two or more active agents so that the combined effect of the ingredients is greater than that obtained when any one ingredient is used alone. Each agent may be in the form of a separate formulation, and the formulations may be the same or different.

[0101] The term “combination therapy” refers to the administration of two or more therapeutic agents or modes of treatment (such as radiotherapy or surgery) to treat the diseases described herein. Such administrations include the simultaneous administration of these therapeutic agents substantially at the same time, such as in a single capsule containing a certain proportion of the active ingredients. Alternatively, such administrations also include the simultaneous administration of each active ingredient in multiple or separate containers (such as tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the required dose before administration. Furthermore, such administrations also include the use of each type of therapeutic agent approximately simultaneously or sequentially at different times. In any case, the treatment regimen will yield the beneficial effects of the combination of pharmaceuticals in the treatment of the conditions or disorders described herein.

[0102] As used herein, “treatment” (or “to treat” or “to treat”) means slowing, interrupting, preventing, alleviating, stopping, reducing or reversing the progression or severity of an existing symptom, condition, situation, or disease.

[0103] As used herein, “prevention” (or “preventive” or “preventive”) includes the suppression of disease or disorder, or the onset or progression of symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive therapy. Generally, in the context of cancer, the term “prevention” (or “preventive”) refers to administering a drug before signs or symptoms of cancer appear, particularly in subjects at risk of cancer.

[0104] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is bound. This term includes not only vectors that function as self-replicating nucleic acid structures, but also vectors that bind to the genome of the host cell to which they are introduced. Some vectors can induce the expression of the nucleic acid to which they are functionally bound. Such vectors are referred to herein as “expression vectors.”

[0105] "Subject / patient / individual sample" refers to a collection of cells or bodily fluids taken from a patient or subject. Sources of tissue or cell samples include solid tissues such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, and puncture samples; bodily fluids such as blood or blood components, cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid; and cells from a subject at any point in time during pregnancy or development. Tissue samples may contain compounds that do not naturally mix with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics.

[0106] The term "freeze-dried formulation" refers to a composition obtained by a freeze-drying process of a liquid formulation. Preferably, it is a solid composition with a moisture content of less than 5%, more preferably less than 3%.

[0107] The term "buffer" refers to a pH buffer capable of maintaining a stable pH in a solution. Preferably, the buffer can maintain the pH of the liquid formulation of the present invention, or the liquid formulation obtained by reconstituting the lyophilized formulation of the present invention, at about 5.5 to 7.5, for example, about 6.0 to 6.5, about 5.5 to 7.2, about 6.0 to 7.0, or about 6.3 to 6.8. Preferably, the buffer is selected from one or more of histidine, citric acid, succinic acid, glutamic acid, phosphoric acid, acetic acid, or salts thereof. The salts include, but are not limited to, histidine hydrochloride or salts formed with acids such as sulfuric acid, and salts formed with alkali metals such as lithium, sodium, or potassium salts, for example, histidine hydrochloride.

[0108] The term "stabilizer" refers to a chemical substance that can maintain the stability of a formulation from a chemical, physical, and / or biological standpoint. Preferably, the stabilizer is selected from one or more carbohydrates and polyols. Carbohydrates may be selected from, but are not limited to, sucrose, trehalose, glucose, lactose, maltose, cyclodextrin, maltodextrin, and dextran. Polyols may be selected from, but are not limited to, mannitol, sorbitol, and xylitol. Carbohydrates or polyols may be in the form of hydrates.

[0109] The term "surfactant" refers to a substance that can significantly reduce the surface tension of a solution system. The concentration of the surfactant in the solution is, for example, about 0.01 mg / mL to 5 mg / mL, e.g., 0.01 to 2, about 0.05 to 1, about 0.05 to 2, about 0.1 to 0.5, about 0.1 to 0.4, about 0.1 to 0.3, or about 0.2 to 0.4 mg / mL. Preferably, the surfactant is a nonionic surfactant and includes, but is not limited to, alkyl poly(ethylene oxide), polysorbates (e.g., polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40), Pluronic, or combinations thereof.

[0110] A “reconstituted” formulation means a liquid formulation obtained by dissolving and / or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution. In this specification, “reconstituted” and “redissolved,” etc., are used interchangeably in terms of formulation.

[0111] As used herein, "w / v" represents "weight / volume," for example, "1%w / v" means 1g / 100mL = 0.01g / mL = 10mg / mL.

[0112] II. Embodiments In one embodiment, the present invention provides a pharmaceutical formulation comprising (i) an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof, (ii) a buffer, (iii) a stabilizer, and / or (iv) a surfactant, wherein the pH of the formulation is about 5.5 to 7.5, preferably about 6.0 to 6.5, and more preferably about 6.5.

[0113] In one embodiment, the antibody-drug conjugate targeting Claudin 18.2 is represented by formula I, [ka] In the formula, L1 is the linker. E is a carbohydrate or carbohydrate derivative, preferably selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc), and N-acetylneuraminic acid (sialic acid). GlcNAc is N-acetylglucosamine, and Fuc is fucose. D is an antitumor drug, preferably exatecan, DXd, MMAE, or DM1. r is 1 to 5, for example 1, 2, 3, 4 or 5, preferably 1 or 2. b is either 0 or 1, In the formula, x is either 1 or 2, and y is from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, or 8, and Preferably, x is 1 and y is 2.

[0114] In some embodiments, E is GalNAc.

[0115] In some embodiments, E is 6-deoxy-2-acetamidogalactose and is linked to L1 via the C atom at the 6th position.

[0116] In some embodiments, the GlcNAc bound to Ab is present at the Asn297 glycosylation sites of the two heavy chains of the antibody.

[0117] In some embodiments, L1 has the following structure,

Chemical formula

[0118] In some embodiments, each L4 is independent, [ka] In the formula, R5 and R6 are independently hydrogen and C1-C 12 Selected from alkyl, and Ar is selected from aryl groups, preferably phenyl.

[0119] In some embodiments, each L4 is independent, [ka] That is the case.

[0120] In some embodiments, -L1- has the following structure. [ka]

[0121] In some embodiments, the average DAR of an antibody-drug conjugate targeting claudin18.2 is 1–15, for example, 2–10, 2–8, or 3–5.

[0122] In one embodiment, an antibody-drug conjugate targeting claudin18.2 is [ka] [ka] Selected from, In the formula, Ab is an antibody or its antigen-binding fragment that targets Claudin18.2. In the formula, q represents the average DAR, In IEX019-02, IEX019-04, and IEX019-05, q ranges from 2 to 5 (e.g., 3 to 5 or 3.5 to 4.5), while in IEX019-03, q ranges from 5 to 11 (e.g., 7 to 9 or 7.5 to 8.5).

[0123] In some embodiments, the heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, preferably the heavy chain constant region of IgG1, for example, the heavy chain constant region of wild-type IgG1. In some embodiments, the antibody light chain constant region LC of the present invention is the lambda or kappa light chain constant region.

[0124] In some specific embodiments of the present invention, the Claudin18.2 antibody of the present invention comprises HCDR1, HCDR2, and HCDR3, each consisting of the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, and / or LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences described in SEQ ID NOs: 6, 7, and 8.

[0125] In some embodiments of the present invention, the Claudin18.2 antibody of the present invention comprises VH and / or VL, where VH consists of the amino acid sequence described in SEQ ID NO: 4 and VL consists of the amino acid sequence described in SEQ ID NO: 9.

[0126] In some embodiments of the present invention, the Claudin18.2 antibody of the present invention comprises a heavy chain and / or a light chain, wherein the heavy chain consists of the amino acid sequence described in SEQ ID NO: 11 and the light chain consists of the amino acid sequence described in SEQ ID NO: 12.

[0127] In one embodiment of the present invention, the amino acid modification described herein includes the substitution, insertion, or deletion of an amino acid. Preferably, the amino acid modification described herein is an amino acid substitution, preferably a conservative substitution. In a preferred embodiment, the amino acid modification described herein occurs in the region outside the CDR (e.g., FR). More preferably, the amino acid modification described herein occurs in the region outside the heavy chain variable region and / or the light chain variable region. In some embodiments, the amino acid modification described herein occurs in the Fc region of the antibody heavy chain constant region.

[0128] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to substituting one amino acid with another amino acid of the same class, for example, substituting an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. In certain embodiments, the substitution occurs in the CDR of the antibody. Generally, the resulting mutant has modified (e.g., improved) certain biological properties (e.g., increased affinity) compared to the parent antibody, and / or substantially retains certain biological properties of the parent antibody. An exemplary substitution mutant is an affinity-mature antibody.

[0129] The Claudin18.2 antibody of the present invention may be an antibody having modified glycosylation. In some embodiments, the antibody is obtained after enzymatic modification of the saccharide chain(s) in vitro (e.g., modification of the saccharide chain(s) by a glycosidase (e.g., endoglycosidase or glycosyltransferase)). In some embodiments, an antibody having modified glycosylation refers to an antibody in which the saccharide chain(s) of the glycosylation site of the antibody is modified from a heterologous N-structure saccharide chain(s) to a mono-structure N saccharide chain(s) having a reactive group (e.g., any reactive group that can react with a linker moiety such as an azide group, ketone group, or alkyne group). In preferred embodiments, the N-glycosylation site is a conserved N-glycosylation site on the Fc domain of an antibody such as Asn297.

[0130] For methods of modifying the glycosylation of antibodies applicable to the present invention, see, for example, PCT / NL2013 / 050744, PCT / EP2016 / 059194, or PCT / EP2017 / 052792, which are incorporated herein by reference in their entirety.

[0131] In a preferred embodiment, the GlcNAc-E(A)x substituent is located at the Asn297 glycosylation site of the two heavy chains of the antibody.

[0132] In some embodiments, L in formula (I) of the present invention is a linker. Any linker known in the art can be used for binding with anti-human CLDN18.2 of the present invention, and preferably the linker can achieve site-directed conjugation of the ADC.

[0133] In some embodiments, the linker applicable to the present invention may be any linker capable of conjugating an antibody to a drug. In some embodiments, the linker may be a linker used in a technique capable of achieving site-directed conjugation.

[0134] In preferred embodiments, the linker of the present invention is an antibody oligosaccharide-bound linker. The term “antibody oligosaccharide-bound linker” as defined herein is any linker that conjugates the antibody to a drug by binding to a reactive group on the saccharide chain (or more) of the glycosylation site of the antibody. The saccharide chain (or more) of the glycosylation site of the antibody is generally a saccharide chain (or more) and is generally modified to convert a heterogeneous saccharide chain (or more) into a single-structure saccharide chain (or more) with a reactive group. The reactive group on the saccharide chain (or more) further binds to the “linker” to achieve site-specific conjugation of the drug and antibody, thus obtaining an antibody-drug conjugate.

[0135] In one embodiment, the “antibody oligosaccharide-bound linker” of the present invention refers in particular to any linker capable of achieving site-specific conjugation with a reactive group on the N-glycosylation site of a conserved N-glycosylation site on the Fc domain of an antibody (e.g., Asn297), for example, the linkers of PCT / NL2013 / 050744 or PCT / EP2021 / 075401, the patent applications of which are incorporated herein by reference in their entirety.

[0136] In one embodiment, the reactive group of the present invention is an azide group, a ketone group, or an alkyne group. In one embodiment, the linker of the present invention is a linker containing an alkyne group. In one embodiment, the reactive group of the present invention is an azide group, a ketone group, or an alkyne group, preferably an azide group, and the linker of the present invention is a linker containing an alkyne group. When referring to such types of linkers in the present invention, since the reactive group reacts with the linker group to form a new group, the group formed after the reaction of the reactive group in the antibody-drug conjugate can also be defined as part of the “linker,” for example, as shown in formula (I) of the present invention.

[0137] Linkers suitable for use in the present invention include, for example, cathepsin-degradable linkers, such as Val-Cit linkers (e.g., vc-PAB), cBu-Cit linkers, and CX linkers; non-cleavable linkers, such as SMCC linkers and MD linkers; acid-sensitive linkers, silyl ether linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, and UV-sensitive linkers such as PC4AP (Antibody-drug conjugates: Recent advances in linker chemistry, Su, Z., Xiao, D., Xie, F., Liu, L., Wang, Y., Fan, S., Li, S. (2021). Antibody-drug conjugates: Recent advances in linker chemistry. Acta Pharmaceutica Sinica B). The linkers suitable for use in the present invention may also be combinations of one or more linkers. For example, a cathepsin-degradable linker can be combined with other types of linkers to form a new linker. Thus, the term "linker" as used herein encompasses a single type of linker or a combination of different types of linkers, insofar as it enables the conjugation of the antibody and drug of the present invention. Accordingly, in one embodiment, suitable linkers for use in the present invention are MC-VC-PAB, vc-PAB, SMCC, or MC-GGFG.

[0138] In formula (I) of the present invention, D can be any antitumor compound, and is not particularly limited as long as it has an antitumor effect and a substituent or substructure that can bind to the linker structure. For example, the antitumor compound may be a pharmaceutically active compound that acts on tumors. Preferably, the antitumor compound is one in which part or all of the linker is cleaved within the tumor cell, the antitumor compound is released, and exerts an antitumor effect. When the linker bound to the drug is cleaved, the antitumor compound is released in its original structure and exerts its original antitumor effect.

[0139] In some embodiments, the antitumor compound may be a cytotoxic or chemotherapeutic agent, such as camptothecin, exatecan (a topoisomerase I inhibitor, exatecan), and Dxd (a novel topoisomerase I inhibitor, exatecan derivative), auristatins such as monomethyl auristatin E (MMAE), or maytansinoids such as the small molecule microtubule inhibitor DM1. The structures in the examples of this application show the structures of representative compounds of these antitumor compounds.

[0140] In some embodiments, the buffer is selected from one or more of histidine, citrate, succinic acid, glutamic acid, phosphoric acid, acetic acid, or salts thereof. Here, the salts of amino acids (histidine, glutamic acid) include, but are not limited to, salts formed by amino acids with hydrochloric acid or sulfuric acid. The salts of phosphoric acid, acetic acid, succinic acid, and citrate include, but are not limited to, alkali metal salts of the corresponding acids, such as lithium, sodium, and potassium salts. Preferably, the histidine salt is histidine hydrochloride. Preferably, the buffer is selected from a combination of histidine and histidine hydrochloride, and histidine.

[0141] In some embodiments, the stabilizer is selected from one or more carbohydrates and polyols, preferably the carbohydrate is selected from, but is not limited to, sucrose, trehalose, glucose, lactose, maltose, cyclodextrin, maltodextrin, and dextran, and the polyol is selected from, but is not limited to, mannitol, sorbitol, and xylitol. The carbohydrate or polyol may be in the form of a hydrate.

[0142] In some embodiments, the stabilizer is selected from sucrose, trehalose, mannitol, or a combination thereof. In some embodiments, the stabilizer is sucrose or trehalose. In some embodiments, the stabilizer is a combination of sucrose and mannitol.

[0143] In some embodiments, The buffer is either a combination of histidine and histidine hydrochloride, or histidine alone. The stabilizers are sucrose, trehalose, or a combination of sucrose and mannitol, and / or The surfactant is polysorbate 80.

[0144] In some embodiments, the concentration of the antibody-drug conjugate targeting claudin 18.2 in the pharmaceutical formulation, or a pharmaceutically acceptable salt or solvate thereof, is about 1 to 200 mg / ml, for example, about 5 to 100 mg / ml, about 10 to 80 mg / ml, about 15 to 60 mg / ml, about 20 to 50 mg / ml, about 20 to 40 mg / ml, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 mg / ml, preferably about 15 to 60 mg / ml, more preferably about 20 to 40 mg / mL.

[0145] In some embodiments, the concentration of the buffer in the pharmaceutical formulation is about 1 to 100 mM, about 5 to 50 mM, about 5 to 30 mM, about 5 to 20 mM, or about 10 to 30 mM, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mM, preferably about 5 to 50 mM, more preferably about 10 to 30 mM.

[0146] In some embodiments, the concentration of the stabilizer in the pharmaceutical formulation is about 10-200 mg / ml, about 20-180 mg / ml, about 30-160 mg / ml, about 40-150 mg / ml, about 50-130 mg / ml, about 50-100 mg / ml, or about 60-90 mg / ml, for example, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 mg / ml, preferably about 50-100 mg / ml, more preferably 60-90 mg / ml. If the stabilizer has two or more components, the concentration of each component can be selected from the following ranges or values: approximately 30-160 mg / ml, approximately 40-150 mg / ml, approximately 50-130 mg / ml, approximately 50-100 mg / ml, approximately 60-90 mg / ml, approximately 30-50 mg / ml, or approximately 10-30 mg / ml, for example, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, or approximately 100 mg / ml.

[0147] In some embodiments, the concentration of the surfactant in the pharmaceutical formulation is about 0.01 to 5 mg / ml, for example 0.01 to 2, about 0.05 to 1, about 0.05 to 2, about 0.1 to 0.5, about 0.1 to 0.4, about 0.1 to 0.3, about 0.2 to 0.4 mg / ml, for example about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7 or about 0.8 mg / ml, preferably about 0.05 to 1 mg / ml, more preferably about 0.1 to 0.3 mg / ml.

[0148] In some embodiments, the pH of the pharmaceutical formulation is approximately 5.5 to 7.5, for example, approximately 6.0 to 6.5, for example, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1 or approximately 7.2, more preferably approximately 6.0 to 6.5.

[0149] In some embodiments, the pharmaceutical formulation is (i) Approximately 20-40 mg / ml of an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof. (ii) Approximately 20 mM buffer solution, (iii) A stabilizer of approximately 60-90 mg / ml, and (iv) Contains approximately 0.1 to 0.3 mg / ml of surfactant.

[0150] In some embodiments, the pharmaceutical formulation is (i) Approximately 20-40 mg / ml of an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof. (ii) A buffer solution of approximately 20 mM, which is a combination of histidine and histidine hydrochloride, or histidine, (iii) A stabilizer of approximately 60-90 mg / ml, which is sucrose, trehalose, or a combination of sucrose and mannitol, and (iv) Contains approximately 0.1-0.3 mg / ml of polysorbate 80, The pH of the pharmaceutical formulation is approximately 6.0 to 6.5, preferably approximately 6.5.

[0151] In some embodiments, the pharmaceutical formulation is (i) Approximately 20 mg / ml of an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof. (ii) A buffer solution of approximately 20 mM, which is a combination of histidine and histidine hydrochloride, or histidine, (iii) A stabilizer of approximately 8% (w / v), which is sucrose, trehalose, or a combination of sucrose and mannitol, and (iv) Contains approximately 0.02% (w / v) of polysorbate 80, The pH of pharmaceutical preparations is approximately 6.5.

[0152] In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation.

[0153] In another embodiment, the present invention provides a lyophilized pharmaceutical formulation comprising an antibody-drug conjugate, the lyophilized pharmaceutical formulation which, after reconstitution, can form or provide the above-described pharmaceutical formulation.

[0154] In some embodiments, lyophilized pharmaceutical formulations are prepared by lyophilizing the above-described pharmaceutical formulations.

[0155] In some embodiments, reconstitution of a lyophilized formulation with a vehicle provides a liquid formulation with a pH of about 5.5 to 7.5, preferably 6.0 to 6.5, for example, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, and about 6.5.

[0156] In another aspect, the present invention provides a lyophilized pharmaceutical formulation comprising an antibody-drug conjugate, which is, (i) an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof in an amount of approximately 1% to 60% (w / w), preferably approximately 15% to 40% (w / w), for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% (w / w), (ii) A buffer solution of approximately 0.5% to 10% (w / w), preferably approximately 2% to 5% (w / w), for example 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, or 9% (w / w), (iii) Stabilizers in an amount of approximately 20% to 90% (w / w), preferably approximately 50% to 85% (w / w), for example 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% (w / w), and / or (iv) comprising about 0.05% to 2% (w / w), preferably about 0.1% to 0.5% (w / w), for example 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, or 1.5% (w / w) of surfactants, Antibody-drug conjugates, buffers, stabilizers, and / or surfactants are as defined herein.

[0157] It should be understood that the range formed by two specific numerical values ​​listed as endpoints herein, or the range formed by a specific numerical value and the endpoint of a range as a new endpoint, is assumed by the present invention as if it were specifically disclosed herein.

[0158] In some embodiments, the lyophilized formulation of the present invention is the formulation disclosed in Example 3. Furthermore, embodiments obtained by varying the concentration values ​​of each component in these formulations by 50%, 40%, 30%, 20%, 10%, or 5%, and by varying the pH by 5%, 4%, 3%, 2%, 1%, or 0.5%, are also included in this application.

[0159] In another embodiment, the present invention provides a liquid pharmaceutical formulation comprising an antibody-drug conjugate, which is obtained after reconstituting the above-mentioned lyophilized pharmaceutical formulation with a vehicle, and whose pH is about 5.5 to 7.5, preferably about 6.0 to 6.5, preferably about 6.5.

[0160] In some embodiments, the liquid pharmaceutical formulation is preferably used for injection or infusion, such as intravenous injection, subcutaneous injection, or intramuscular injection.

[0161] In another embodiment, the present invention provides a delivery device comprising a pharmaceutical formulation, a lyophilized pharmaceutical formulation, or a liquid pharmaceutical formulation of the present invention.

[0162] In another aspect, the present invention provides the use of the formulations of the present invention (the above-mentioned pharmaceutical formulations, lyophilized pharmaceutical formulations, or liquid pharmaceutical formulations) in the manufacture of pharmaceuticals, preferably the pharmaceuticals being used for the prevention or treatment of tumors. More preferably, the tumor is cancer, such as epithelial carcinoma or gastrointestinal carcinoma, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.

[0163] Unless otherwise specified, if the formulation of the present invention is a liquid, it should be understood that it further includes, but is not limited to, purified water such as ultrapure water, water for injection, sterile water, and redistilled water, a vehicle, particularly a vehicle for injection. Vehicles that can reconstitute the lyophilized formulation of the present invention include, but are not limited to, purified water such as ultrapure water, water for injection, sterile water, redistilled water, physiological saline, Ringer's solution, glucose injection, etc.

[0164] In some embodiments, the present invention provides pre-filled syringes for intravenous, subcutaneous, or intramuscular injection, comprising the above-described pharmaceutical formulations of the present invention, lyophilized pharmaceutical formulations, or liquid pharmaceutical formulations.

[0165] In another aspect, the present invention provides a method for preparing a pharmaceutical formulation according to the present invention, the method comprising the following steps: i. The present invention provides an isolated and purified antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which may optionally be added to an ultrafiltration centrifuge tube, centrifuged and concentrated, ii. A buffer solution, preferably an aqueous solution, is prepared, and preferably the type and concentration of the buffer solution and stabilizer, and the pH of the solution are as defined in the above-mentioned pharmaceutical formulation. iii. Ultrafiltration is performed, and the antibody-drug conjugate from step i is replaced with the solution from step ii. iv. Adjust the concentration of the substituted antibody-drug conjugate or its pharmaceutically acceptable salt or solvate to the concentration of the above-mentioned pharmaceutical preparation. v. Add a surfactant or a solution thereof, preferably an aqueous solution thereof, to bring the final concentration of the surfactant to the concentration defined in the above pharmaceutical formulation, and vi. Optionally, sterilize and filter the solution from step v.

[0166] In another embodiment, the present invention further provides a method for preparing a lyophilized pharmaceutical formulation of the present invention, in addition to steps i-v and any step vi, the method further comprising the following steps: vii. Freeze-dry the solution from step v or vi to obtain a lyophilized pharmaceutical preparation.

[0167] In another embodiment, the present invention further provides a pharmaceutical combination or a pharmaceutical combination product, The pharmaceutical combination comprises the pharmaceutical formulation of the present invention, a lyophilized pharmaceutical formulation, or a liquid pharmaceutical formulation, and one or more other therapeutic agents (for example, the therapeutic agents include chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (for example, immune checkpoint inhibitors or agonists)).

[0168] In another embodiment, the present invention provides a kit of parts comprising a pharmaceutical formulation, a lyophilized pharmaceutical formulation, or a liquid pharmaceutical formulation, or a pharmaceutical combination of the present invention, preferably in the form of a drug administration unit. Thus, the administration unit may be provided according to an administration regimen or drug administration interval.

[0169] In one embodiment, the kit of parts of the present invention includes the following in the same package: - A first container containing the formulation of the present invention, - A second container containing a pharmaceutical composition including other therapeutic agents(s).

[0170] In another aspect, the present invention provides a method for preventing or treating a tumor (e.g., cancer) in a subject, comprising administering an effective amount of the formulation, combination of pharmaceuticals, or kit of the present invention to the subject.

[0171] In some embodiments, a patient having a tumor (e.g., cancer) has increased levels of CLDN18.2 (e.g., nucleic acid or protein). In some embodiments, the patient's tumor cells express CLDN18.2, for example, moderately, and preferably highly.

[0172] In some embodiments, tumors, such as cancer, include solid tumors, hematological malignancies, and metastatic lesions. In one embodiment, an example of a solid tumor includes a malignant tumor. Cancer can be early-stage, intermediate-stage, advanced, or metastatic.

[0173] In certain embodiments, the formulations of the present invention can kill tumor cells and / or inhibit the proliferation of tumor cells, for example, tumor cells expressing CLDN18.2, for example, gastrointestinal tumor cells, for example, gastric cancer cells or pancreatic cancer cells or colon cancer cells or colorectal cancer cells.

[0174] In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface, and in some embodiments, the target cells are CHO cells or 293 cells expressing CLDN18.2 (e.g., CHO-S cells or HEK293 cells). In some embodiments, the target cells are cancer cells expressing CLDN18.2, such as cells that spontaneously express CLDN18.2, cells that express CLDN18.2 after artificial transfection, or cells with increased CLDN18.2 expression levels after artificial transfection, such as gastric cancer cells, pancreatic cancer cell lines, colon cancer, or colorectal cancer cell lines expressing CLDN18.2. In some embodiments, the target cells are cell lines with moderate hCLDN18.2 expression levels, such as NUGC-4 or SNU620. In some embodiments, the target cells are cell lines with high hCLDN18.2 expression levels, such as DAN-G cells overexpressing hCLDN18.2.

[0175] In some embodiments, the tumor is a tumor immune evasion mechanism.

[0176] In some embodiments, the tumor is cancer, such as an epithelial tumor, such as a gastrointestinal tumor, such as an epithelial carcinoma, or gastrointestinal cancer, such as gastric cancer, gastroesophageal junction cancer, pancreatic cancer, colorectal cancer, or colon cancer.

[0177] The subjects may be mammals, such as primates, preferably evolved primates, such as humans (for example, individuals who have or are at risk of developing one of the diseases described herein). In one embodiment, the subject has or is at risk of developing one of the diseases described herein (for example, cancer). In certain embodiments, the subject is receiving or has received other treatments such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0178] In another aspect, the present invention provides the use of formulations, pharmaceutical combinations, or kits of the present invention in the manufacture or production of pharmaceuticals for the use described herein, for example, for the manufacture or production of pharmaceuticals for the prevention or treatment of the relevant diseases or disorders described herein.

[0179] In some embodiments, the formulations, drug combinations, or kits of the present invention can delay the onset of disorders and / or symptoms associated with disorders.

[0180] In some embodiments, the formulations of the present invention may also be administered in combination with one or more other therapies, such as therapeutic modes and / or other therapeutic agents, for use as described herein, for example, to prevent and / or treat the relevant diseases or disorders described herein.

[0181] In some embodiments, the treatment mode includes surgery, radiotherapy, or local or focused irradiation.

[0182] In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0183] Other exemplary antibodies include antibodies that specifically bind to immune checkpoints.

[0184] The combination therapy of the present invention encompasses combination administration (for example, including two or more therapeutic agents in the same or separate formulations) and separate administration. In the latter case, the pharmaceutical formulation of the present invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or drugs.

[0185] The composition can also be administered topically via an embedded membrane, sponge, or other suitable material that absorbs or encapsulates the desired molecule. In certain embodiments, when an implantable device is used, the device can be implanted in any suitable tissue or organ and can deliver the target molecule by diffusion, time-release bolus, or continuous administration.

[0186] In another aspect, the present invention provides the formulations of the present invention described above for use in therapy and / or as pharmaceuticals, for example, for use as described herein, for example, for the prevention or treatment of related diseases or disorders as described herein.

[0187] These and other aspects and embodiments of the present invention are shown in the drawings (brief description of the drawings) and the following detailed description of the invention, and are illustrated in the following examples. Any or all of the features described above and throughout the invention can be combined in various embodiments of the invention. The following examples further illustrate the invention. However, these examples are provided for illustrative purposes only and not limiting, and it should be understood that various modifications are possible. [Examples]

[0188] Example 1.1 Synthesis of IEX019 ADC molecule The anti-CLDN18.2 monoclonal antibody HB37A6 is prepared according to CN202010570517.X (corresponding to PCT / CN2021 / 100870). Based on HB37A6, ADCs conjugated with various small molecule compounds were further designed and synthesized. The specific process is as follows:

[0189] Example 1.1.1 Preparation of IEX019-02 1) Preparation of compound 6 [ka] Under an N2 atmosphere, chlorosulfonyl isocyanate (CSI) (0.87 mL, 1.4 g, 10 mmol), Et3N (2.8 mL, 2.0 g, 20 mmol), and 2-(2-aminoethoxy)ethanol (1.2 mL, 1.26 g, 12 mmol) were added to a solution of BCN-OH (5, 1.5 g, 10 mmol) in DCM (150 mL). The mixture was stirred for 10 minutes and quenched with saturated NH4Cl aqueous solution (150 mL). After separation, the aqueous layer was extracted with DCM (150 mL). The combined organic layers were dried and concentrated with (Na2SO4). The residue was purified by column chromatography. Product 6 (2.06 g, 5.72 mmol, 57%) was obtained as a pale yellow, concentrated oil. 1 H NMR(400MHz,CDCl3)δ(ppm)6.0(bs,1H),4.28(d,J=8.2Hz,2H),3.78-3.73(m,2H),3.66-3.61(m,2H),3.61-3.55(m ,2H),3.34(t,J=4.9Hz,2H),2.37-2.15(m,6H),1.64-1.48(m,2H),1.40(quintet,J=8.7Hz,1H),1.05-0.92(m,2H).

[0190] 2) Preparation of compound 7 [ka] CSI (11 μL, 18 mg, 0.13 mmol) was added to a stirred solution of 6 (47 mg, 0.13 mmol) in DCM (10 mL). After 30 minutes, a solution of Et3N (91 μL, 66 mg, 0.65 mmol) and diethanolamine (16 mg, 0.16 mmol) in DMF (0.5 mL) was added. After 30 minutes, p-nitrophenyl chloroformate (52 mg, 0.26 mmol) and Et3N (54 μL, 39 mg, 0.39 mmol) were added. After a further 4.5 hours, the reaction mixture was concentrated. The residue was purified by gradient column chromatography (33 → 66% Â / heptane (1% AcOH)) to obtain 7 (88 mg, 0.098 mmol, 75%) as a colorless oil. 1 H NMR(400MHz,CDCl3)δ(ppm)8.28-8.23(m,4H),7.42-7.35(m,4H),4.52(t,J=5.4Hz,4H),4.30(d,J=8.3Hz,2H),4.27-4.22(m,2H),3.86(t,J=5.3Hz, 4H),3.69-3.65(m,2H),3.64-3.59(m,2H),3.30-3.22(m,2H),2.34-2.14( m, 6H), 1.62-1.46 (m, 2H), 1.38 (quintet, J=8.7Hz, 1H), 1.04-0.92 (m, 2H).

[0191] 3) Preparation of compound 9 [ka] Compound 8 (163 mg, 240 μmol) was added to a mixture of exatecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in dry DMP (0.9 mL). After 20 hours, the reaction mixture was diluted in 9 mL of DCM and purified by gradient column chromatography (0 → 40% MeOH / DCM) to obtain compound 9 (155 mg, 159 μmol, 68%). LCMS(ESI+)C 55 H 54 FN6O 10 + (M+H) + Calculated value: 977.39, Measured value: 977.80.

[0192] 4) Preparation of Compound 1 A solution of Et3N (73 mg, 101 μL, 0.72 mmol) and compound 7 (65 mg, 72 μmol) in DMF (1.4 mL) was added to a solution of compound 9 (155 mg, 159 μmol) in DMF (1.6 mL). The reaction mixture was stirred for 24 hours, diluted with DCM (20 mL), and purified by gradient column chromatography (0 → 40% MeOH / DCM) to obtain compound 1 (94 mg, 44 μmol, 28%) as a pale yellow solid. LCMS(ESI+)C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + Calculated value: 1066.88, Measured value: 1067.12.

[0193] 5) HB37A6 was enzymatically reconstituted into HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2. HB37A6 was expressed in HEK293 cells and purified. The obtained HB37A6 (16.4 mg / mL) was incubated with EndoSH (1% w / w) as described in PCT / EP2017 / 052792 to obtain trimmed HB37A6 with -GlcNAc or GlcNAc(Fuc) at the Asn297 position. The trimmed HB37A6 was incubated with the enzyme His-TnGalNAcT (4.5% w / w) disclosed in PCT / EP2016 / 059194 and 6-azido-GalNAc-UDP (25 eq (equivalents) compared to the antibody) disclosed in PCT / EP2016 / 059194 (in a solution of histidine (20 mM) + NaCl (150 mM) containing 6 mM MnCl2) for 16 hours at 30°C.

[0194] Subsequently, the resulting incubation mixture was purified using a 50 mL protA column (Hitrap Mabselect Sure, GE, 11-0034-95). The incubation mixture obtained above was introduced into the column and washed with TBS + 0.2% Triton and TBS. Next, the column was eluted with 0.1 M acetate buffer (pH 2.9) and neutralized with 2.5 M Tris-HCl (pH 7.2). After dialyzing three times with PBS, the obtained (modified) glycosylated antibody was concentrated to 32.6 mg / mL using a Vivaspin Turbo 15 ultrafiltration system (Sartorius).

[0195] The obtained glycosylated antibody was analyzed by mass spectrometry, with the main steps being as follows: The glycosylated antibody was treated with IdeS prior to mass spectrometry, and the Fc / 2 fragment was analyzed. 20 μg of (modified) glycosylated antibody solution was incubated with 10 μL total volume of IdeS (Fabricator®) (1.25 U / μL) in PBS (pH 6.6) at 37°C for 1 hour. The sample was diluted to 80 μL and analyzed by JEOL AccuTOF (ESI-TOF), and the deconvoluted spectrum was obtained using Magtran software. Mass spectrometry of the IdeS digested sample revealed that the main product corresponds to the obtained HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2, with an observed mass of 24,330.4.

[0196] Therefore, the above results demonstrate that HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 was obtained in which the GlcNAc of Asn297 in both heavy chains was replaced with 6-azide-GalNAc (substituted at position 4).

[0197] 6) Preparation of HB37A6-SYNtecan E complex (IEX019-02) The bioconjugate IEX019-02 of the present invention was prepared by conjugating compound 1 (linker payload) as a linker complex to azide-modified HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 as a biomolecule. Accordingly, to a solution of HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 (10.8 mL, 350 mg, 32.6 mg / ml, PBS pH 7.4), PBS pH 7.4 (808 μL), 1,2-propylene glycol (11.3 mL), and compound 1 (350 μL, 40 mM DMF solution) were added. The reaction mixture was incubated overnight at room temperature, then filtered and dialyzed to PBS pH 7.4. Charcoal (350 mg) was added, and residual free drugs were removed by rotation overnight at room temperature. The charcoal was removed by centrifugation and filtration. ADCs were purified using an AKTA Purifier-10 (GE Healthcare) equipped with a Superdex200 26 / 600 SEC (GE Healthcare) column.

[0198] Mass spectrometry of the IdeS digestion sample revealed two main products corresponding to the resulting ADC, namely the HB37A6-SYNtecan E complex. The first peak, with an observed mass of 26469 Da (calculated mass of 26465 Da), corresponds to the bound Fc / 2 fragment (a blocked lactone form with twice the payload). The second peak, with an observed mass of 26499 Da (calculated mass of 26501 Da), corresponds to the bound Fc / 2s fragment (an open carboxylate form with twice the payload).

[0199] The obtained structure is as follows. The concentration, DAR value, and SEC purity of the ADC product were determined by UV, SEC, RP-HPLC, and LC-MS. The monomer purity detected by SE-HPLC was over 99%, and the concentration was 6.12 mg / ml. [ka] In the formula, q represents the mean DAR, for example, 3-5, 3.2-4.8, or 3.5-4.5 (or 3.52 if measured in Table 1). In the formula, Ab is HB37A6.

[0200] Example 1.1.2 Preparation of IEX019-03 [ka] In the formula, q represents the mean DAR, for example, 5-11, 6-10, 7-9, or 7.5-8.5 (or 7.9 if measured in Table 1). In the formula, Ab is HB37A6.

[0201] (a) Add the reducing agent solution (TCEP (Sigma, C4706), dissolved in water) to the HB37A6 solution (antibody HB37A6 dissolved in PBS buffer (Thermo, 10010023)), shake the reaction mixture in a shaker for 2-4 hours, (i) The optimal concentration of HB37A6 was 5–10 mg / mL. (ii) The optimal molar ratio of TCEP / mAb was 4.5 to 6.5. (iii) The optimal temperature for the reaction was 20–37°C. (iv) The optimal pH value for the reaction was generally between 6.5 and 8.0.

[0202] (b) Excess linker toxin MC-GGFG-DXD (purchased from Levena Biopharma, SET0218, structure shown below) was dissolved in DMSO and reacted with the thiol group of the antibody reduced in step (a). The reaction mixture was shaken in a shaker for 1-2 hours. (i) The optimal molar ratio of DXD / mAb is 10.0 to 12.0. (ii) The optimal temperature for the reaction is 20-37°C. A rough ADC was obtained.

[0203] (c) The obtained crude ADC was purified by spin desalting, ultrafiltration, or dialysis to obtain the final ADC product IEX019-03.

[0204] (d) The ADC product was detected by HIC, LC-MS and SEC HPLC, and the average DAR value and SEC purity were determined.

Chemical formula

[0205] Example 1.1.3 Preparation of IEX019-04

Chemical formula

[0206] This molecule was prepared according to the following method.

[0207] (a) A reducing agent solution (TCEP (Sigma, C4706), dissolved in water) was added to a solution of antibody HB37A6 (the antibody HB37A6 was dissolved in PB buffer). After the addition was completed, the reaction mixture was shaken on a shaker for 2 - 4 hours. (i) The optimal concentration of antibody HB37A6 was 5 - 10 mg / mL. (ii) The optimal molar ratio of TCEP / mAb was 1.9 - 2.7. (iii) The optimal temperature for the reduction reaction was 20 - 37 °C. (iv) The optimal pH value for the reaction was generally 6.5 - 8.0.

[0208] (b) An excess of a linker payload MC-VC-PAB-MMAE (purchased from Levena biopharma, SET0221) having a reactive group (maleimide-linked drug) was dissolved in an organic solvent DMSO and reacted with the reduced thiol groups generated in step (a). The reaction mixture was shaken on a shaker for 1 - 2 hours. (i) The optimal molar ratio of MC-VC-PAB-MMAE / mAb was 8.0 - 10.0. (ii) The optimal temperature for the binding reaction was 20 - 37 °C.

[0209] (c) After the binding reaction was complete, acetylcysteine ​​solution was added to stop the reaction in step (b). After mixing, the mixture was incubated at 20-25°C for 5-15 minutes.

[0210] (d) The obtained crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain the final ADC product IEX019-04.

[0211] (e) The ADC product was detected by HIC and SEC-HPLC, and the average DAR value and SEC purity were determined. [ka]

[0212] Example 1.1.4 Preparation of IEX019-05 [ka] In the formula, q represents the mean DAR, for example, 3-5, 3.2-4.8, or 3.0-4.5 (3.3 if measured in Table 1). In the formula, Ab is HB37A6,

[0213] This molecule is prepared according to the following method:

[0214] (a) Add the linker payload SMCC-DM1 (purchased from Levena Biopharma, SET0101, dissolved in an organic solvent such as DMSO) to the antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer), and after addition, shake the reaction mixture in a shaker for 2-5 hours. (i) The optimal concentration of antibody HB37A6 is 5-10 mg / mL. (ii) The optimal molar ratio of linker payload / mAb is 5.5 to 6.5. (iii) The optimal pH value for the reaction is generally 6.5 to 8.0. (iv) The optimal temperature for the reaction is 20-37°C. Crude ADC product obtained,

[0215] (b) The obtained crude ADC product was purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IEX019-05.

[0216] (c) The average DAR value and SEC purity of the ADC product were determined using ultraviolet spectrophotometry and SEC high performance liquid chromatography.

Chemical formula

[0217] Example 1.1.5 Preparation of IEX019-06 The preparation process was the same as that of IEX019-02, except that the HB37A6 monoclonal antibody was replaced with the reference antibody IgG.

[0218] Example 1.1.6 Preparation of IEX019-07 The preparation process was the same as that of IEX019-04, except that the HB37A6 monoclonal antibody was replaced with the reference antibody IgG.

[0219] All the information on monoclonal antibodies and ADCs is summarized in Table 1.

Table 2

[0220] Example 1.2 Cell Binding Experiment of IEX019 Molecule To test whether small molecule conjugation changes the binding characteristics of the IEX019-01 monoclonal antibody to target cells, the DAN-G cell line (hCLDN18.2 negative) and DAN-G-hCLDN18.2 cell line (hCLDN18.2 overexpression) prepared in Example 1 of CN202010570517.X were used, and the affinity of IEX019-01 and IEX019-02 to the target was detected by flow cytometry (FACS). The specific experimental method is as follows.

[0221] Count the test cells (CHO-hCLDN18.2) and calculate 1 × 10⁻⁶ 6 The solution was diluted to cells / ml and added to a U-bottom 96-well plate at a rate of 100 μl / well. The cell culture medium was removed by centrifugation at 500 g for 5 minutes. Molecules IEX019-01, IEX019-02, and the reference molecule IEX019-06 were added to the U-plate, and cells were resuspended in 100 μl portions per well. The initial concentration of the above molecules was 900 nM, and then sequentially diluted threefold to a total of 10 concentrations. The mixture was placed on ice for 30 minutes. The supernatant was removed by centrifugation at 500 g for 5 minutes. The cells were washed once with PBS. 100 μl of goat anti-human Fc PE-labeled secondary antibody (SouthernBiotech, J2815-5H87B) was added to each well. The mixture was incubated on ice in the dark for 30 minutes. The mixture was centrifuged at 500 g for 5 minutes, and the supernatant was removed. The cells were washed once with PBS. Cells were resuspended in 50 μl of 1×PBS and detected by FACS. Experimental data were analyzed using GraphPad Prism software.

[0222] IEX019-01 and IEX019-02 did not bind to non-target cells DAN-G, but showed strong affinity for DANG-hCLDN18.2, indicating that antibody binding is dependent on the expression specificity of the target, and that exatecan conjugation does not affect antibody binding. At the same time, the reference molecule IEX019-06 (negative control using IgG as a monoclonal antibody, IgG conjugated to exatecan toxin using the same technique) did not bind to target cells (Figure 1).

[0223] Example 1.3 Endocytosis experiment of IEX019 molecule Potent endocytosis is one of the key characteristics of ADC drugs. When an ADC binds to an antigen on the cell membrane surface, the ADC antigen complex enters the cell via endocytosis and kills the target cell. Therefore, the endocytosis efficiency of ADCs is one of the important indicators that determine the tumor inhibitory effect.

[0224] To test the endocytosis efficiency of antibodies after binding to small molecule compounds, endocytosis of various IEX019 molecules on DANG-hCLDN18.2 cells was examined by flow cytometry. After digesting the DANG-hCLDN18.2 cells, the cell density was adjusted. Cells were placed in 1*10⁶ well plates in a 96-well plate. 5 Cells were seeded in individual wells and centrifuged at 500g for 3 minutes. The supernatant was discarded, 100 μl of the tested molecule was taken and the cells were resuspended (molecular concentration 50 nM), and five replicate experiments were set up for each sample (endocytosis times: 0 hours, 1 hour, 2 hours, 3 hours, 4 hours). The mixture was placed on ice and incubated for 1 hour. After 1 hour, the mixture was centrifuged at 500g for 3 minutes. The supernatant was discarded. Each well was washed twice with 200 μl of FACS buffer (1% FBS, 1XPBS). One set of samples was taken, transferred to a new 96-well plate, and incubated at 37°C for 4 hours. The remaining samples were kept incubated on ice. The previous procedure was repeated. The samples were incubated at 37°C for 3 hours, 2 hours, 1 hour, and 0 hours, respectively. After the specified incubation time, the mixture was centrifuged at 500g for 3 minutes. The supernatant was discarded. 100 μl of anti-hFC-PE antibody (SouthernBiotech), diluted 1:400, was added to each well. The mixture was incubated on ice in the dark for 30 minutes. After secondary antibody incubation, the mixture was centrifuged at 500 g for 3 minutes. The supernatant was discarded. Each well was washed twice with 200 μl of FACS buffer. Finally, the cells were resuspended in 100 μl of PBS and detected.

[0225] The experimental results are shown in Figure 2, where 0 hours of incubation at 37°C represents the zero point of endocytosis, and after 2 hours of incubation, all molecules reached the maximum amount of endocytosis (approximately 60%), indicating that the ADC molecule designed and synthesized based on IEX019-01 maintains potent endocytic ability after binding to tumor cells, consistent with the monoclonal antibody.

[0226] Example 1.4 In vitro cell-killing effect of the IEX019 molecule The effect of ADCs on cell viability was tested using the CellTiter-Glo (Promega, G9242) assay kit in various cell lines expressing hCLDN8.2 at varying levels (Table 2). [Table 3]

[0227] After digesting the used cells with EDTA / trypsin, the density was adjusted. The cells were evenly seeded in 96-well plates (Table 3). Diluted samples of IEX019 molecule at specific concentrations (IEX019-02, IEX019-03, IEX019-04, initial dilution concentration 100 nM, 3-fold dilution) were added, and wells without IEX019 molecule were used as controls. The plates were placed in a 37°C incubator for 5 days. After 5 days, 100 μl of CellTiter-Glo detection reagent was added to each well, followed by incubation at room temperature for 30 minutes and detection with a microplate reader. Relative cell viability (relative cell viability = sample / control * 100%) was calculated, and the curves were fitted using Graph Pad Prism 8.0.

[0228] As shown in Figure 3, cell line killing by the ADC molecule depended on the surface expression level of hCLDN18.2. In hCLDN8.2-negative DANG cells, the IEX019 molecule had no significant effect on cell viability (Figure 3A), while in cell lines with moderate expression levels (NUGC-4, SNU620), the IEX019 molecule showed some degree of cell killing (Figure 3B). In high-expression cell lines DAN-G-hCLDN18.2, the IEX019 molecule showed a significant killing effect in all cases (Figure 3C). The results demonstrated that the IEX019 molecule possesses excellent selectivity and efficacy. [Table 4]

[0229] Example 1.5: Bystander lethality In the synthesis process of ADC drugs, small molecule compounds bind to antibodies via a cleavable linker. After entering the cell membrane via endocytosis, the linker is cleaved, releasing the small molecule and killing the target cell. After the target cell dies, the small molecule compound is released from the target cell into the intercellular space, further killing non-target cells within a certain range. This effect is known as bystander killing. Because cells within a tumor can have significantly different levels of target expression (tumor heterogeneity), bystander killing is important for effectively killing tumor cells and suppressing tumor growth.

[0230] In this invention, non-target cells DAN-G and target cells DANG-hCLDN18.2 were used to test the bystander-killing effect of the IEX019 molecule.

[0231] After digesting the used cells with EDTA / trypsin, the density was adjusted. A 6-well cell culture plate was prepared, and 7.5 × 10⁶ cells were added. 4100 DANG cells and 100 DANG-hCLDN18.2 cells were added. The two cell types were co-incubated. 200 μl of test samples (IgG (SEQ ID NO: 13, SEQ ID NO: 14), IEX019-02, IEX019-05, IEX019-06, exatecan (Macklin, E881532)) were added to each well at a final concentration of 50 nM, and each sample was repeated three times. The cell culture plates were placed in a 37°C incubator for 5 days. After 5 days, the culture supernatant was discarded. After washing with PBS, the cells were digested with trypsin-EDTA. All digested cells were collected and transferred to a 96-well plate. Following the antibody incubation protocol of flow cytometry technology, the cells were incubated with primary antibody (IEX019-01, 100 nM) and secondary antibody (anti-hFc-PE, Southern Biotech) at 4°C for 1 hour and 0.5 hours, respectively. After washing with PBS, the viability stain (Thermo, L34964) was diluted 1:1,000 and 100 μl was added to each well, and incubated at 4°C for 20 minutes. After washing with PBS, the cells were resuspended in 100 μl of PBS and detected. Populations within each sample were distinguished using viability staining; IEX019-01 negative (i.e., hCLDN18.2 negative) populations were DAN-G cells, and IEX019-01 positive (i.e., hCLDN18.2 positive) cells were DAN-G-hCLDN18.2. The number of each cell type in each sample was counted, and the relative cell viability of each cell type was calculated according to the following formula, and the curve was fitted in Graph Pad Prism 8.0.

[0232] Relative viability of DANG cells = Number of DNAG cells in the sample group / Number of DNAG cells in the IgG group * 100%

[0233] Relative viability of DANG-hCLDN18.2 cells = Number of DNAG-18.2 cells in the sample group / Number of DNAG-18.2 cells in the IgG group * 100%

[0234] The cell viability of the IgG group was set to 100%.

[0235] As shown in Figure 4, the negative control sample IEX019-06 showed no killing effect against either type of cell. The IEX019-05 molecule was only able to kill DANG-hCLDN8.2 cells and had no effect on DANG cells because it was unable to bind the DM1 toxin to the IEX019-01 monoclonal antibody via a non-cleaving linker and kill surrounding non-target cells. Only IEX019-02 exhibited a significant bystander killing effect and was able to kill both target and non-target cells.

[0236] Example 1.6: Antitumor effect of the IEX019 molecule in a DAN-G-hCLDN18.2 mouse xenograft tumor model To demonstrate the in vivo efficacy of the IEX019 molecule, the antitumor effect of the antibody molecule of the present invention was tested using CB-17-SCID mice inoculated with DANG-hCLDN18.2 cells. SPF-grade female CB-17-SCID mice (14-17g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for the experiment, with certificate number NO.110011201108225246.

[0237] DANG-hCLDN18.2 cells were subcultured as usual for subsequent in vivo experiments. DANG-hCLDN18.2 cells were collected by centrifugation and dispersed in PBS (1×) to a cell concentration of 3×10⁶. 6 A cell suspension was prepared at a concentration of cells / ml. A DANG-hCLDN18.2 tumor-bearing mouse model was established by subcutaneously inoculating 0.2 ml of the cell suspension into the right flank of CB-17 SCID mice on day 0.

[0238] Five days after tumor cell inoculation, the tumor volume was 50.16–136.68 mm². 3 All mice were grouped in a curved manner (6 mice per group). Dosage and administration methods are shown in Table 4. Administration was performed on day 5 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 5A and 5B, until monitoring ended after 92 days.

[0239] The relative tumor growth inhibition rate (TGI%) was calculated 50 days after vaccination, using the following formula.

[0240] TGI% = 100% * (tumor volume in the control group - tumor volume in the treatment group) / (tumor volume in the control group - tumor volume in the control group before administration).

[0241] Tumor volume measurement: The maximum long axis (L) and maximum width (W) of the tumor were measured with calipers, and the tumor volume was calculated according to the following formula.

[0242] V=L*W 2 / 2. Weight was measured using an electronic balance. [Table 5]

[0243] The results of tumor growth inhibition are shown in Table 5 and Figure 5A. At 50 days post-inoculation, the tumor growth inhibition of a single dose of IEX019-02 reached 103.60% compared to the IEX019-01 monoclonal antibody, which was significantly superior to IEX019-03 and IEX019-04, which showed tumor growth inhibition of 93.70% and 35.20%, respectively. At 82 days post-inoculation, complete tumor regression was observed in 100% of the mice in the IEX019-02 group. Simultaneously, when the body weight of the mice was measured, no significant difference was observed, as shown in Figure 5B. [Table 6]

[0244] Example 1.7: Antitumor effect of the IEX019 molecule in a NUGC-4 mouse xenograft tumor model To demonstrate the in vivo efficacy of the IEX019 molecule, the antitumor effect of the antibody molecule of the present invention was tested using CB-17-SCID mice inoculated with NUGC-4 cells.

[0245] The experiment used SPF-grade female CB-17-SCID mice (14-17g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), with certificate number NO.110011201109348141.

[0246] NUGC-4 cells were subcultured as usual for subsequent in vivo experiments. NUGC-4 cells were collected by centrifugation and dispersed in PBS (1×) to a cell concentration of 3×10⁶. 7 A cell suspension was prepared at a concentration of cells / ml. A NUGC-4 tumor-bearing mouse model was established by subcutaneously inoculating 0.2 ml of the cell suspension into the right flank of CB-17 SCID mice on day 0.

[0247] Five days after tumor cell inoculation, the tumor volume was 72.25–140.50 mm². 3 All mice were grouped in a curved manner (6 mice per group). Dosage and administration methods are shown in Table 6. Administration was performed on day 5 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 6A and 6B, until monitoring ended after 40 days.

[0248] The relative tumor growth inhibition rate (TGI%) was calculated 33 days after vaccination, using the following formula.

[0249] TGI% = 100% * (tumor volume in the control group - tumor volume in the treatment group) / (tumor volume in the control group - tumor volume in the control group before administration).

[0250] Tumor volume measurement: The maximum long axis (L) and maximum width (W) of the tumor were determined using calipers, and the tumor volume was calculated according to the following formula.

[0251] V=L*W 2 / 2. Weight was measured using an electronic balance. [Table 7]

[0252] The results of tumor growth inhibition are shown in Table 7 and Figure 6A. The tumor growth inhibition rates after single administration of IEX019-02 and IEX019-03 on day 33 after inoculation were 80.04% and 54.31%, respectively, compared to the negative control IEX019-06. Simultaneously, the body weight of the mice was measured, and as shown in Figure 6B, no significant difference was observed in the body weight of the mice. [Table 8]

[0253] Example 1.8: Antitumor effect of IEX019 molecule in SNU620 mouse xenograft tumor model To demonstrate the in vivo efficacy of the IEX019 molecule, the antitumor effect of the antibody molecule of the present invention was tested using CB-17-SCID mice inoculated with SNU620 cells. SPF-grade female CB-17-SCID mice (18-20g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for the experiment, with certificate number NO.110011211102179364.

[0254] SNU620 cells were subcultured as usual for subsequent in vivo experiments. SNU620 cells were collected by centrifugation and dispersed in PBS (1×) to a cell concentration of 3×10⁶. 7 A cell suspension was prepared at a concentration of cells / ml. A mouse model carrying SNU620 tumors was established by subcutaneously inoculating 0.2 ml of the cell suspension into the right flank of CB-17 SCID mice on day 0.

[0255] Seven days after tumor cell inoculation, the tumor volume was 58.1–117.3 mm². 3 All mice were grouped in a curved manner (6 mice per group). Dosage and administration methods are shown in Table 8. Administration was performed on day 7 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 7A and 7B, until monitoring ended at day 39.

[0256] The relative tumor growth inhibition rate (TGI%) was calculated on the 39th day after vaccination, using the following formula.

[0257] TGI% = 100% * (tumor volume in the control group - tumor volume in the treatment group) / (tumor volume in the control group - tumor volume in the control group before administration).

[0258] Tumor volume measurement: The maximum long axis (L) and maximum width (W) of the tumor were measured with calipers, and the tumor volume was calculated according to the following formula.

[0259] V=L*W 2 / 2. Weight was measured using an electronic balance. [Table 9]

[0260] The results of tumor growth inhibition are shown in Table 9 and Figure 7A. The tumor growth inhibition rate after a single dose of IEX019-02 10 mg / kg on day 39 post-inoculation was 143.77% compared to hIgG, and tumors completely regressed in 100% of mice. There was no significant difference in body weight between the two treatment groups compared to the hIgG control group (Figure 7B). [Table 10]

[0261] Example 2: Formulation study of the IEX019-02 formulation 1. Overview The objective of this experiment was to develop a long-term stable IEX019-02 formulation. The development of the formulation consisted of two steps: 1) screening of pH buffers (stored for 4 weeks under stress conditions at 40°C), and 2) screening of auxiliary materials, including placing liquid samples under stress conditions at 25°C and 40°C for 4 weeks, followed by 5 freeze-thaw cycles and 3 days of vibration (150 rpm, 25°C). Lyophilized samples were stored at 40°C for 2, 4, and 12 weeks. Analytical methods included DSC, appearance, pH, ADC molecular concentration, insoluble particles (HIAC), SE-HPLC, iCIEF, Caliper-R / NR, DAR value, free agent, reconstitution time, and binding activity.

[0262] 2. Overview of Analytical Methods for Pharmaceutical Formulations 2.1 Exterior The appearance of the samples, including transparency, color, and visible particles, was examined under a black and white background using a YB-2 transparency detector.

[0263] 2.2 pH value The pH of the sample was measured using a pH meter with a glass electrode. Before first use each day, the pH meter was calibrated using three different standard buffer solutions (pH 4.01, 7.00, and 9.21). The calibration gradient was in the range of 95.0% to 105.0%, and the zero drift was in the range of -60.0mV to +60.0mV.

[0264] 2.3 Molecular Concentration of ADC The absorbance of ADC at 280 nm and 365 nm was detected using Nanodrop or solo-VPE, and the molecular concentration of ADC was calculated according to the following formula: CF is the correction factor, AWV is the absorbance at 365 nm, and EC is the extinction coefficient (1.52 Au * mL * mg - 1 * cm - 1).

number

[0265] 2.4 Insoluble particles (HIAC) Insoluble particles were sized and counted under a laminar flow hood using a HIAC particle analyzer (HIAC 9703+). 0.45 mL of sample was tested per run, and four runs were performed consecutively. The results of the first run were discarded, and the average number of insoluble particles per milliliter in the last three runs was reported (insoluble particle size ≥ 2 / 10 / 25 μm).

[0266] 2.5 Purity of Size Exclusion Chromatography (SE-HPLC) SE-HPLC is a purity analysis method that separates proteins based on their size. The SE-HPLC analysis procedure was performed as follows: Detection was performed on an Agilent 1260 HPLC system equipped with a TSKgel G3000SWXL column (7.8 × 300 mm, particle size 5 μm) and a UV detector (detection wavelength: 280 nm). The sampler and column oven temperatures were set to 5 ± 3 °C and 25 ± 3 °C, respectively. The mobile phase consisted of 0.078 M potassium dihydrogen phosphate, 0.122 M dipotassium hydrogen phosphate trihydrate, 0.15 M potassium chloride, and 15% (v / v) IPA (pH 7.0 ± 0.1), with a flow rate of 0.8 mL / min. 100 μg of each sample was injected into an HPLC vial. The detection wavelength was set to 280 nm, and the analysis time was 20 minutes. Finally, all raw data were processed using CDS software.

[0267] 2.6 Drug-to-Antibody Ratio (DAR) The DAR analysis procedure was as follows: Detection was performed using a Waters Ultra Performance Liquid Chromatograph UPLC system equipped with a Waters / ACQUITY UPLC Protein BEH C4 column, 300 Å, column (2.1 × 150 mm, particle size 1.7 μm), and a UV detector (detection wavelength: 280 nm). The sampler and autosampler temperatures were set to 5 ± 3 °C and 70 ± 1 °C, respectively. Mobile phase A was 0.1% trifluoroacetic acid in water containing 5% IPA, and mobile phase B was 0.1% trifluoroacetic acid in acetonitrile containing 5% IPA. The flow rate was 0.25 mL / min. 100 μg of each sample was injected into an HPLC vial. The detection wavelength was set to 280 nm, and the analysis time was 45 minutes. Finally, all raw data were processed using Empower software.

[0268] 2.7 Free Agents The procedure for free drug analysis was as follows: Detection was performed using an Agilent Technologies 1260 system equipped with an Agilent Eclipse Plus C18 column (4.6 × 100 mm, particle size 3.5 μm) and a UV detector (detection wavelength: 365 nm). The sampler and column temperatures were set to 5 ± 3 °C and 50 ± 1 °C, respectively. Mobile phase A was 0.1% trifluoroacetic acid in water, and mobile phase B was 0.085% trifluoroacetic acid in acetonitrile. The flow rate was 1.0 mL / min. 40 μL of each sample was injected into an HPLC vial. The detection wavelength was set to 365 nm, and the run time was 18 minutes. Finally, all raw data were processed using Empower3 software.

[0269] 2.8 Charge variant iCIEF The sample was diluted to 2.0 mg / ml with ultrapure water, and 20 μL of the diluted sample was mixed with 80 μL of a pre-mixed solution to prepare a loading mixture with a final concentration of 0.4 mg / mL. The pre-mixed solution consisted of 4.0 μL of amphoteric electrolyte 3-10, 35 μL of 1% methylcellulose, 0.5 μL of isoelectric focusing marker with pI 7.05, 0.5 μL of isoelectric focusing marker with pI 9.91, 37.5 μL of 8M urea solution, 1.0 μL of 500 mM arginine solution, and 1.5 μL of 3.3% HAc solution. Detection had to be completed within 48 hours of mixing the sample with the pre-mixed solution. The loading solution was analyzed using an iCE3 capillary isoelectric focusing analyzer with an FC-coated full-column imaging capillary. After separation, the raw data was processed using Water / Empower FR5.0.

[0270] 2.9 Purity of Caliper-NR / R in Reducing / Non-Reducing Capillary Gel Electrophoresis Non-reducing caliper-SDS was analyzed using a SCIEX / PA800 Plus instrument equipped with a PDA detector (diode array detector). The sample was diluted to 2.0 mg / mL, 50 μL of the sample was placed in a 1.5 mL centrifuge tube, 5 μL of NEM (N-ethylmaleimide) was added to the tube, and 45 μL of SDS sample buffer (PB-CA (citrate phosphate buffer) containing 0.8% SDS) was added. The sample was thoroughly mixed, centrifuged, and incubated at 70°C for 10 minutes. The capillary right-end injection method (effective capillary separation length 20.2 cm) was used. The detection wavelength was 220 nm. After continuous injection for 20 seconds using a forward voltage of -5 kV, separation was performed for 35 minutes with a forward voltage of +15 kV.

[0271] Reduced Caliper-SDS was analyzed using a SCIEX / PA800 Plus instrument equipped with a PDA detector. The sample was diluted to 2.0 mg / mL. 50 μL of the sample was placed in a 1.5 mL centrifuge tube, and 45 μL of pH 6.5 sample buffer (PB-CA (phosphate citrate buffer) containing 0.8% SDS) and 5 μL of BME (β-mercaptoethanol) were added. The sample was thoroughly mixed and centrifuged, then left at 70°C for 10 minutes, and then centrifuged at 13,000 rpm for approximately 1 minute. The left-end capillary injection method (effective capillary separation length was 20.2 cm) was used. The detection wavelength was 220 nm. After continuous injection for 20 seconds using a reverse voltage of -5 kV, separation was performed for 35 minutes using a reverse voltage of -15 kV.

[0272] 2.10 Binding activity Serially diluted reference, control, and test samples were added to a 96-well high-binding plate, and the products were bound to the antigen coated on the plate. After washing, peroxidase-labeled goat anti-human IgG Fcγ detection antibody (Jackson, 109-035-098#) was added to the plate. The detection antibody can bind to the captured ADC product. After the final wash, a TMB (3,3',5,5'-tetramethylbenzidine) substrate solution was added to the plate. TMB reacts specifically with peroxide in the presence of peroxidase to produce a monochromatic signal, the intensity of which is proportional to the amount of ADC product bound on the plate. After the color reaction was complete, absorbance values ​​from 450 nm to 620 nm were read using a microplate reader.

[0273] Using a four-parameter fitted regression model in SoftMax Pro software, the EC50 values ​​for the reference, control, and test samples within the same plate were obtained, and the relative binding activity between the control and test samples was calculated using the following formula.

[0274] Relative binding activity of the control or test sample = EC50 of the reference / EC50 of the control or test sample × 100%.

[0275] 2.11 Reconfiguration time A 10ml syringe was used to collect more than 5.2ml of sterile water for injection, and air was removed so that the final volume of sterile water in the syringe was 5.2ml. The syringe was inserted through the center of the stopper. After adding the sterile water along the wall of the bottle, the timer was pressed. The aluminum cap on the bottle opening was grasped with the hand. The wrist was rotated slightly clockwise. If no particles were observed within 10 minutes, the timer was pressed again, and that time was used as the reconstruction time.

[0276] 2.12 DSC A DSC (Dynamic Spectrometer) is a thermal analyzer that measures the temperature difference between a test sample and a reference sample during heating. This experiment was performed using a MicroCal PEAQ DSC (Malvern, SYS12907). Before analysis, the protein sample was first diluted to 1 mg / mL using a reference buffer. 400 μL of each reference buffer was added to the odd-numbered wells of a 96-well plate, and 400 μL of the sample was added to the even-numbered wells of the same plate. The experimental parameters were set so that the scan temperature increased from 10°C to 95°C at a rate of 90°C / hour. Thermal gram analysis was performed using MicroCal PEAQ DSC automated data analysis software.

[0277] 2.13 Detection of the glass transition temperature (Tg') The glass transition temperature (Tg') of protein formulations was measured using a TA2000 DSC. 20 μL of sample was placed in an aluminum sample tray, and both the sample tray and an empty sample tray (see reference) were placed inside the DSC instrument. Corresponding parameters, including heating / cooling rate, target heating / cooling temperature, and platform retention time after reaching the target temperature (see table below), were set on the control panel. Once the settings were complete, data sampling was initiated. During this process, mDSC patterns were recorded, and the glass transition temperature (Tg') was obtained by Universal Analysis. [Table 11]

[0278] 2.14 Decay temperature (Tc) The decay temperature (Tc) was measured using a Lyostat5 LED freeze-drying microscope. When testing a sample, the sample holder was placed on the temperature control block, and the side door of the sample cell was closed to ensure the sample holder was centered on the temperature control block. A 16 mm diameter quartz glass piece was placed on the sample holder with a vacuum pen and brought into contact with the temperature control block. Next, a 70 μm thick metal gasket was grasped with tweezers and placed on the quartz slide so as to be coaxial with the center of the quartz sheet. A certain amount (typically 2 μL) of test sample was pipetted and dropped into the center of the quartz sheet. A 13 mm diameter coverslip was carefully placed on the metal gasket with a vacuum pen so that the edge of the sample was positioned above the light-transmitting hole of the temperature control block, allowing for easy visualization of the sample. The lid was rotated back on the platform, and the nitrogen outlet pipe was secured on top of the lid to prevent frost. Then, the corresponding parameters (see table below), including the heating / cooling rate, the target heating / cooling temperature, the platform holding time after reaching the target temperature, and the frequency of image acquisition, were set on the control panel. Once the setup was complete, data sampling began. During freeze-drying, the sublimation front moved from the outer edge of the sample to the center. The front of the sublimation front remained frozen, while the rear remained dry. When the sample temperature rose above the decay temperature (Tc), decay occurred. The photographs used in the experiment were carefully reviewed, starting with those taken at lower temperatures. If small voids appeared in the photographs, it indicated microstructural decay, and the corresponding temperature was considered the decay temperature. [Table 12]

[0279] 3. Screening of pH buffer 3.1 Objective of the experiment The purpose of this experiment is to screen for the optimal pH of the buffer system for IEX019-02 at a concentration of 40 mg / mL.

[0280] 3.2 Experimental Protocol As shown in Table 10, three buffer systems were studied in eight experimental groups at a concentration of 40 mg / mL IEX019-02.

[0281] The stability of three different pH buffer systems in eight experimental groups was evaluated over four weeks under high-temperature conditions (40°C). The test parameters performed in this study included DSC, appearance, pH, ADC molecular concentration, SE-HPLC, Caliper-NR / R, free agents, DAR value, and iCIEF. [Table 13]

[0282] 3.3 Sample Preparation The IEX019-02 stock solution (10 mg / mL ADC, 20 mM histidine solution, 8% (w / v) sucrose, pH 6.0) was concentrated in an ultrafiltration tube, and then replaced with systems of 20 mM citrate buffer (pH 5.0, 6.0), 20 mM succinate buffer (pH 5.0, 5.5, 6.0), and 20 mM histidine buffer (pH 5.5, 6.0, 6.5), respectively. The concentration of IEX019-02 was adjusted to approximately 40 mg / mL. The solutions were filtered through a 0.22 μm PES filter in a biosafety cabinet to obtain samples F1-F8 shown in Table 10. The samples were filled into 2 mL vials at a filling volume of 1 mL / vial. Immediately after filling, a stopper was attached to the vial and the cap was closed. The samples were placed under different storage conditions according to Table 10, and at least one vial was placed at each sampling point for each formulation.

[0283] 3.4 Results and Discussion The results of pH buffer screening for the formulations of IEX019-02 are summarized in Table 11-20.

[0284] 3.4.1 DSC As shown in Table 11 and Figure 8, the Tm start values ​​for all samples in the eight pH buffer systems (F1-F8) were above 58°C, indicating that the molecules possess good structural stability. [Table 14]

[0285] 3.4.2 Appearance At T0, the samples were pale yellow to pale milky liquids containing small amounts of protein particles in eight pH buffer systems (F1-F8). After being left at 40°C for one or two weeks, numerous visible particles and turbidity were observed in F1-F2 (citric acid buffer system, pH 5.0 and pH 6.0), F6 (histidine / histidine hydrochloride buffer system, pH 5.5), and F3-F4 (succinic acid / sodium succinate buffer system, pH 5.0 and pH 5.5). Small amounts of particles were observed in F5 (succinic acid / sodium succinate buffer system, pH 6.0) and F7-F8 (histidine / histidine hydrochloride buffer system, pH 6.3 and 6.5), and the samples were clear. After being left at 40°C for four weeks, numerous particles were present in all samples, but the amount of particles in F1-F4 was relatively high.

[0286] 3.4.3 pH value As shown in Table 12, with the exception of the histidine 6.0 group (F7), no significant change in pH value was observed in the other pH buffer systems (F1-F6, F8) after being left at 40°C for 4 weeks compared to T0. [Table 15]

[0287] 3.4.4 Molecular Concentration of IEX019-02 As shown in Table 13, no significant change in IEX019-02 concentration was observed after storing samples from eight pH buffer systems (F1-F8) at 40°C for four weeks, compared to T0. [Table 16]

[0288] 3.4.5 Purity of Size Exclusion Chromatography SE-HPLC As shown in Table 14, compared to T0, SE-HPLC monomers in the eight pH buffer systems (F1-F8) decreased significantly after being left at 40°C for 1, 2, and 4 weeks. At the end of week 1, SE-HPLC monomers decreased by 1.7% to 23.1%, and SE-HPLC monomers in F6-F8 (histidine buffer systems, pH 5.5, pH 6.3, pH 6.5) decreased by 1.7% to 2.6%. At the end of week 2, SE-HPLC monomers decreased by 8.2% to 30.2%, and SE-HPLC monomers in F6-F8 (histidine buffer systems, pH 5.5, pH 6.3, pH 6.5) decreased by 8.2% to 10.5%. At the end of week 4, SE-HPLC monomers decreased by 9.4% to 56.2%, and SE-HPLC monomers in F6 to F8 (histidine buffer systems, pH 5.5, pH 6.3, pH 6.5) decreased by 9.4% to 10.8%, which was a lower rate of decrease than in the other pH buffer systems. Therefore, histidine buffer is preferred. [Table 17]

[0289] 3.4.6 Charge variant iCIEF As shown in Table 15, the iCIEF main peaks of the eight pH buffer systems (F1-F8) decreased by 11.6% to 21.8% compared to T0 after being left at 40°C for 4 weeks. The iCIEF main peaks of F7 (histidine buffer, pH 6.3), F8 (histidine buffer, pH 6.5), and F5 (succinate buffer, pH 6.0) decreased by 11.6% to 17.1%, which was lower than the other pH buffer systems (iCIEF main peak decreased by 18.6% to 21.8%). Therefore, F7, F8, and F5 performed better. [Table 18]

[0290] 3.4.7 Purity of reduced capillary electrophoresis Caliper-R As shown in Table 16, compared to T0, the purity of Caliper-R for the eight pH buffer systems (F1-F8) decreased by 1.0% to 4.6% after being left at 40°C for 4 weeks. The purity of Caliper-R for F5 (succinate buffer system, pH 6.0) and F6-F8 (histidine buffer systems, pH 5.5, pH 6.3, pH 6.5) decreased by 1.0%, which was lower than the other pH buffer systems (Caliper-R purity decreased by 1.4% to 4.6%). Therefore, F5, F6, F7, and F8 performed best. [Table 19]

[0291] 3.4.8 Purity of non-reducing capillary electrophoresis (Caliper-NR) As shown in Table 17, compared to T0, the purity of Caliper-NR for eight pH buffer systems (F1-F8) decreased by 3.6% to 8.5% after being left at 40°C for 4 weeks. The purity of Caliper-NR in F2 (citrate buffer system, pH 6.0) decreased by 3.6%, which was lower than the purity of the other pH buffer systems (Caliper-NR purity decreased by 4.0% to 8.5%). [Table 20]

[0292] 3.4.9 Free agents As shown in Table 18, compared to T0, the free drug content in the eight pH buffer systems (F1-F8) increased by 25.8% to 34.4% mol / mol after being left at 40°C for 4 weeks. The free drug content in F8 (histidine buffer system, pH 6.5) increased by 25.8% mol / mol, which was lower than that of the other pH buffer systems (free drug content increased by 29.2% to 34.4% mol / mol). Therefore, F8 performed better. [Table 21]

[0293] 3.4.10 Drug-to-Antibody Ratio (DAR) As shown in Table 19, compared to T0, the DAR values ​​of the eight pH buffer systems (F1-F8) decreased by 1.84 to 2.95 after being left at 40°C for 4 weeks. The DAR value of F8 (histidine buffer system, pH 6.5) decreased by 1.84, which was lower than that of the other pH buffer systems (DAR values ​​decreased by 2.19 to 2.95). Therefore, F8 performed better. [Table 22]

[0294] 3.4.11 Summary Based on the DSC and stability results at 40°C, the F8 formulation (20 mM histidine buffer, pH 6.5) sample was ultimately selected for the next auxiliary material screening because it exhibited a high Tm onset value, good appearance under stable conditions, relatively high purity in size exclusion chromatography, high stability of charge isomers, minimal decrease in drug-to-antibody ratio, and low levels of free drug.

[0295] 4. Auxiliary material screening experiment 4.1 Objective of the experiment The objective of this study is to screen for optimal auxiliary materials for a histidine buffer system and a 40 mg / mL IEX019-02 concentration.

[0296] 4.2 Experimental Protocol The formulation design and sampling plan for the auxiliary material screening experiment are shown in Tables 20 and 21. Lyophilized samples of three formulations with four types of auxiliary materials were incubated under high-temperature conditions for 12 weeks. Liquid samples were incubated under high-temperature conditions (40°C and 25°C for 4 weeks), freeze-thaw cycles (5 cycles from room temperature to -70°C), and vibration conditions (150 rpm, vibration at 25°C for 3 days). The optimal auxiliary material was selected according to the stability of the three formulations. Test items included appearance, pH value, ADC molecular concentration, SE-HPLC, iCIEF, Caliper-R / NR, drug-to-antibody ratio, free drug, decay temperature Tc, glass transition temperature Tg', DSC, insoluble particles (HIAC method), moisture content and binding activity (ELISA) of the lyophilized samples. [Table 23] [Table 24]

[0297] 4.3 Sample Preparation According to the stock solution composition and experimental design, the IEX019-02 stock solution (40 mg / mL ADC, 20 mM histidine solution, 8% (w / v) sucrose, pH 6.5) was taken and replaced by ultrafiltration with 20 mM histidine solution, 8.8% trehalose dihydrate (w / v) (pH 6.5) solution, and 20 mM histidine buffer, 2% sucrose (w / v), and 4% mannitol (w / v) (pH 6.5) solution. At this point, the concentration of IEX019-02 exceeded 40 mg / mL. According to Table 20, IEX019-02 was diluted to 40 mg / mL by adding the corresponding ultrafiltration buffer, and then 5% polysorbate 80 was added to a concentration of 0.02% (w / v) to obtain Fe01 to Fe03. After preparation was complete, samples of each formulation were filtered through a 0.22 μm PES filter and filled into 20 mL vials and 6 mL vials with filling volumes of 5 mL / vial and 3 mL / vial, respectively. After filling, the 20 mL vials were sealed with a half-stopper and immediately lyophilized according to the procedure described in Table 22, while the 6 mL vials were sealed and capped. The samples were stored under different storage conditions according to Table 21, and at least one vial for each formulation was placed at each sampling point. [Table 25]

[0298] 4.4 Results and Discussion The results of the auxiliary material screening experiment are summarized in Tables 23-35.

[0299] 4.4.1 Sample Tc, Tg', and Tm values As shown in Table 23, the Tc of all three formulations (Fe01-Fe03) was higher than -21°C. The Tc of Fe01 and Fe02 were similar, while the Tc of Fe03 was higher at -9.2°C. The Tg' varied considerably, being -28.3°C, -26.6°C, and -32.8°C, respectively. The Tm onset temperatures of all three formulations (Fe01-Fe03) were higher than 67.0°C, and the Tm values ​​were higher than 79°C, indicating that these three formulations have good stability. [Table 26]

[0300] 4.4.2 Reconstruction time and moisture content results As shown in Table 24, the reconstitution times of the freeze-dried samples of the three formulations (Fe01-Fe03) differed significantly, with Fe01 having a relatively short reconstitution time. There was no significant change in the moisture content of the three groups during the stabilization process, remaining below 0.6%. [Table 27]

[0301] 4.4.3 Exterior As shown in Table 25, liquid samples of the three formulations (Fe01-Fe03) were pale yellow and pale milky liquids with no visible particles at T0. After 3 days of vibration, 5 freeze-thaw cycles, and 4 weeks at 25°C, the three formulations (Fe01-Fe03) were substantially free of visible particles or had no visible particles at all. However, visible particles appeared after 2 weeks and 4 weeks at 40°C. The lyophilized samples were completely powdery cake-like and pale yellow before reconstruction, but showed almost no visible particles after reconstruction. [Table 28]

[0302] 4.4.4 Molecular concentration of ADC As shown in Table 26, compared to T0, there was no significant change in the ADC molecular concentration of the liquid samples of the three formulations (Fe01-Fe03) after storage at 40°C or 25°C for 4 weeks, followed by 5 cycles of freeze-thaw or 3 days of vibration at 25°C. No significant change in the ADC molecular concentration was observed when the lyophilized samples were left at 40°C for 12 weeks. [Table 29]

[0303] 4.4.5 pH value As shown in Table 27, after placing liquid samples of the three formulations (Fe01-Fe03) at 40°C or 25°C for 4 weeks, followed by 5 freeze-thaw cycles or 3 days of vibration at 25°C, there was no significant change in pH compared to T0. No significant change in pH was observed even after lyophilized samples were placed at 40°C for 12 weeks. [Table 30]

[0304] 4.4.6 Insoluble particles (HIAC) As shown in Table 28, during five freeze-thaw cycles, there was no significant change in the insoluble particles of the liquid samples of the three formulations (Fe01-Fe03) compared to T0. However, after 3 days of vibration at 25°C, the insoluble particles of Fe03 increased significantly, while the remaining samples (Fe01-Fe02) showed no significant change. When the three formulations (Fe01-Fe03) were placed at a high temperature of 40°C for 2 weeks, the particle size increased significantly. The insoluble particles of the samples of the three formulations (Fe01-Fe03) increased slightly after freeze-drying. After freeze-drying, when the samples were placed at a high temperature of 40°C for 2 weeks, 4 weeks, and 12 weeks, there was no significant change in the insoluble particles of Fe01, but the insoluble particles of Fe02 and Fe03 increased significantly. [Table 31]

[0305] 4.4.7 Purity of Size Exclusion Chromatography (SE-HPLC) As shown in Table 29, compared to T0, no significant changes were observed in the SE-HPLC monomers after 5 cycles of freeze-thawing or 3 days of oscillation at 25°C for liquid samples of the three formulations (Fe01-Fe03). However, the SE-HPLC purity decreased significantly after being left at 40°C or 25°C for 4 weeks. No significant changes were observed in the SE-HPLC purity of the three formulation samples after lyophilization. After leaving the lyophilized samples at 40°C for 2, 4, and 12 weeks, the SE-HPLC purity of Fe01, Fe02, and Fe03 decreased by 1.3%, 2.0%, and 5.4%, respectively. [Table 32]

[0306] 4.4.8 Charge variant iCIEF As shown in Table 30, compared to T0, there was no significant change in the iCIEF main peaks of the three formulations after 4 weeks at 25°C, followed by 5 freeze-thaw cycles or 3 days of vibration at 25°C. After 4 weeks at 40°C, the iCIEF main peaks of the three formulations (Fe01-Fe03) decreased by 9.8%-11.8%. After lyophilization, no significant change was observed in the iCIEF main peaks of the three formulations (Fe01-Fe03). When the lyophilized samples were left at 40°C for 2, 4, and 12 weeks, the iCIEF main peaks of the three formulations (Fe01-Fe03) decreased by 2.0%-3.1% after 12 weeks. [Table 33]

[0307] 4.4.9 Purity of reduced capillary electrophoresis Caliper-R As shown in Table 31, compared to T0, there was no significant change in the purity of Caliper-R of the three formulations (Fe01-Fe03) after being left at 25°C for 4 weeks, followed by 5 freeze-thaw cycles and 3 days of vibration at 25°C. However, when the liquid samples were left at 40°C for 4 weeks, and when the lyophilized samples were left at 40°C for 12 weeks, the purity of Caliper-R decreased slightly. [Table 34]

[0308] 4.4.10 Purity of non-reducing capillary electrophoresis (Caliper-NR) As shown in Table 32, compared to T0, there was no significant change in Caliper-NR purity after liquid samples of the three formulations (Fe01-Fe03) were left at 25°C for 4 weeks, followed by 5 cycles of freeze-thawing or 3 days of vibration at 25°C. After being left at 40°C for 4 weeks, the Caliper-NR purity of the three formulations (Fe01-Fe03) decreased by 5.3%-5.5%. After being left at 40°C for 12 weeks, the Caliper-NR purity of the lyophilized samples of the three formulations (Fe01-Fe03) decreased by 0.9%-2.9%. [Table 35]

[0309] 4.4.11 Free agents As shown in Table 33, compared to T0, there was no significant change in the free agents of the three formulations (Fe01-Fe03) after the liquid samples were left at 25°C for 2 weeks followed by 5 cycles of freeze-thawing or 3 days of vibration at 25°C. However, after 4 weeks at 25°C, the free agents increased by 1.4%-4.5%, and after 2 weeks at 40°C, the free agents increased by 26.3%-33.7%, or the free agents of the three formulations (Fe01-Fe03) increased significantly by 41.6%-50.1% after 4 weeks. When lyophilized samples were left at 40°C for 2, 4, and 12 weeks, no significant change in the free agents was observed after 4 weeks, and after 12 weeks, the free agents of the three formulations (Fe01-Fe03) increased by 0.9%-4.2%. [Table 36]

[0310] 4.4.12 Drug-to-Antibody Ratio (DAR) As shown in Table 34, compared to T0, there was no significant change in the DAR values ​​of the three formulations (Fe01-Fe03) after the liquid samples were left at 25°C for 4 weeks, followed by 5 cycles of freeze-thawing, or 3 days of vibration at 25°C. However, after being left at 40°C for 2 weeks, the DAR values ​​decreased by 0.95-1.12, and the DAR values ​​of the three formulations (Fe01-Fe03) decreased by 1.51-1.74 after 4 weeks. When the freeze-dried samples were left at 40°C for 2, 4, and 12 weeks, no significant change in the DAR values ​​was observed after 4 weeks, and after 12 weeks, the DAR values ​​of the three formulations (Fe01-Fe03) decreased by 0.41-0.56. [Table 37]

[0311] 4.4.13 Binding activity As shown in Table 35, the binding activity of the samples was tested at weeks 2 and 4. Compared to T0, there was no significant change in activity when the liquid sample was left at 25°C for 2 weeks, or when the freeze-dried T0 sample was left at 40°C for 2 and 4 weeks after freeze-drying. [Table 38]

[0312] 4.4.14 Summary Based on the overall results and the analysis described above, Fe01 (20 mM histidine / histidine hydrochloride, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5) was selected as the formulation ingredient because its stability was superior to that of the other formulations.

[0313] 5. Checking the ingredients 5.1 Objective of the experiment Lyophilized samples were prepared using formulations screened during the auxiliary material screening stage, and the final formulations and lyophilization process were confirmed by long-term (2-8°C), accelerated (25±2°C), and high-temperature (40±2°C) stability experiments with the lyophilized samples.

[0314] 5.2 Experimental Protocol Stability experiments were conducted on the DP formulations (final product formulations) obtained from the screening of auxiliary materials under long-term (2-8°C), accelerated (25±2°C), and high-temperature (40±2°C) conditions to investigate the stability of the lyophilized formulations and confirm the final formulations. The stability protocol is shown in Table 36. [Table 39]

[0315] 5.3 Sample Preparation The 20 mg / mL stock solution was divided into 20 mL vials at 5.6 mL / bottle. The 40 mg / mL solution was also divided into 20 mL vials at 5.6 mL / bottle. After partially stopping the vials, they were freeze-dried, and the freeze-drying process is shown in Table 37. As described in Table 36, the freeze-dried DP was placed under different test conditions (long-term experiment at 5°C, accelerated experiment at 25°C, and high-temperature experiment at 40°C) and sampled for detection at the specified times. Three vials of DP were placed at each time point. [Table 40]

[0316] 5.4 Results and Discussion The results of stability experiments under long-term, accelerated, and high-temperature conditions are summarized in Table 38.

[0317] The results showed that after investigating samples with IEX019-02 concentrations of 20 mg / mL and 40 mg / mL at 2–8°C for 12 weeks, the appearance before reconstitution showed no significant change compared to T0 before reconstitution, and the appearance after reconstitution also showed no significant change compared to T0. Furthermore, no significant changes were observed in pH, ADC concentration, reconstitution time, moisture content, drug-to-antibody ratio, purity by size exclusion chromatography, purity by non-reducing capillary electrophoresis, purity by reducing capillary electrophoresis, insoluble particles, charge variants, and free drugs, indicating that the samples are stable under 2–8°C conditions. However, the reconstitution time for the 20 mg / mL IEX019-02 concentration sample was shorter than that for the 40 mg / mL concentration sample.

[0318] Samples with an IEX019-02 concentration of 20 mg / mL were investigated under accelerated conditions at 25°C for 12 weeks. Compared to T0, there were no significant changes in appearance before and after reconstitution. No significant changes were observed in pH, protein concentration, reconstitution time, moisture content, drug-to-antibody ratio, purity by size exclusion chromatography, purity by non-reducing capillary electrophoresis, purity by reducing capillary electrophoresis, insoluble particles, charge variants, or free drugs.

[0319] Samples with IEX019-02 concentrations of 20 mg / mL and 40 mg / mL were investigated under high-temperature conditions of 40°C for 4 weeks. The appearance before and after reconstitution showed no significant changes compared to T0, and no significant changes were observed in pH, protein concentration, moisture content, drug-to-antibody ratio, purity by size exclusion chromatography, purity by non-reducing capillary electrophoresis, purity by reducing capillary electrophoresis, insoluble particles, charge variants, or free drugs. However, the reconstitution time for the IEX019-02 20 mg / mL sample was shorter than that for the ADC molecule 40 mg / mL sample. [Table 41-1] [Table 41-2] [Table 41-3]

[0320] In summary, the formulations obtained through screening showed good stability during long-term storage (2-8°C) for 12 weeks, and the major qualitative properties shown in accelerated and high-temperature stability experiments did not change significantly compared to T0. Therefore, the formulation of "20 mg / ml ADC, 20 mM histidine buffer, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5" was confirmed to be appropriate.

[0321] Sequence information: [Table 42-1] [Table 42-2]

Claims

1. A pharmaceutical formulation comprising (i) an antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof, (ii) a buffer, (iii) a stabilizer, and / or (iv) a surfactant, The pharmaceutical preparation wherein the pH of the pharmaceutical preparation is about 5.5 to 7.5, preferably about 6.0 to 6.5, and more preferably about 6.

5.

2. The antibody-drug conjugate targeting Claudin 18.2 is 【Chemistry 1-1】 [Chemistry 1-2] Selected from, In the formula, Ab is an antibody or antigen-binding fragment that targets Claudin 18.

2. In the formula, q represents the average DAR, The pharmaceutical formulation according to claim 1, wherein in IEX019-02, IEX019-04, and IEX019-05, q is between 2 and 5 (e.g., 3 to 5 or 3.5 to 4.5), and in IEX019-03, q is between 5 and 11 (e.g., 7 to 9 or 7.5 to 8.5).

3. The antibody-drug conjugate targeting Claudin 18.2 is 【Chemistry 2-1】 【Chemistry 2-2】 Selected from, In the formula, Ab is an antibody or antigen-binding fragment that targets Claudin 18.2, and In equation I-A, y is an integer of 1 or 2. The pharmaceutical preparation according to claim 1, wherein in formulas I-B, I-C, or I-D, y is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

4. The antibody or antigen-binding fragment targeting Claudin 18.2 comprises HCDR1, HCDR2, and HCDR3, each consisting of the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3, each consisting of the amino acid sequences described in SEQ ID NOs: 6, 7, and 8, respectively. Preferably, the antibody or antigen-binding fragment targeting Claudin 18.2 includes a heavy chain variable region and / or a light chain variable region, the heavy chain variable region is (i) containing or consisting of the amino acid sequence described in Sequence ID No. 4, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in Sequence ID No. 4, or comprising such an amino acid sequence, (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No. 4, wherein the amino acid modifications do not occur in the CDR, and / or The aforementioned light chain variable region is (i) containing or consisting of the amino acid sequence described in Sequence ID No. 9, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in Sequence ID No. 9, or comprising such an amino acid sequence, (iii) An amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No. 9, or comprising such an amino acid sequence, preferably such amino acid modifications do not occur in the CDR. A pharmaceutical preparation according to claim 2 or 3.

5. The antibody or antigen-binding fragment targeting Claudin 18.2 comprises a heavy chain and a light chain, the heavy chain being (i) containing or consisting of the amino acid sequence described in Sequence ID No. 11, or (ii) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in Sequence ID No. 11, or comprising such an amino acid sequence, (iii) an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No. 11, or comprising such an amino acid sequence and / or The aforementioned light chain is (i) containing or consisting of the amino acid sequence described in Sequence ID No. 12, (ii) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in Sequence ID No. 12, or comprising such an amino acid sequence, (iii) A pharmaceutical preparation according to any one of claims 2 to 4, comprising or consisting of an amino acid sequence having one or more (preferably 20 or 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence described in Sequence ID No.

12.

6. The pharmaceutical formulation according to any one of claims 1 to 5, comprising about 1 to 200 mg / ml, preferably 15 to 60 mg / ml, and more preferably about 20 to 40 mg / ml, of the antibody-drug conjugate targeting Claudin 18.2 or the pharmaceutically acceptable salt or solvate thereof.

7. The pharmaceutical formulation according to any one of claims 1 to 6, wherein the pharmaceutical formulation comprises about 1 to 100 mM, preferably about 5 to 50 mM, and more preferably about 10 to 30 mM of the buffer solution.

8. The pharmaceutical formulation according to any one of claims 1 to 7, wherein the pharmaceutical formulation comprises about 10 to 200 mg / ml, preferably about 50 to 100 mg / ml, and more preferably about 60 to 90 mg / ml of the stabilizer.

9. The pharmaceutical formulation according to any one of claims 1 to 8, wherein the pharmaceutical formulation contains the surfactant in an amount of about 0.01 to 5 mg / ml, preferably about 0.05 to 1 mg / ml, and more preferably about 0.1 to 0.3 mg / ml.

10. The buffer solution is selected from one or more of histidine, citrate, succinic acid, glutamic acid, phosphoric acid, acetic acid, or salts thereof. Preferably, the buffer is a combination of histidine and histidine hydrochloride, or histidine, according to any one of claims 1 to 9.

11. The aforementioned stabilizer is selected from one or more carbohydrates and polyols. Preferably, the carbohydrate is selected from sucrose, trehalose, glucose, lactose, maltose, cyclodextrin, maltodextrin, and dextran, and the polyol is selected from mannitol, sorbitol, and xylitol. More preferably, the stabilizer is selected from sucrose, trehalose, mannitol, or a combination thereof, as described in any one of claims 1 to 10.

12. The surfactant is selected from alkyl poly(ethylene oxide), polysorbate, pluronic, or a combination thereof. Preferably, the surfactant is selected from polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40, as described in any one of claims 1 to 11.

13. The aforementioned pharmaceutical preparation is (i) an antibody-drug conjugate targeting Claudin 18.2, or a pharmaceutically acceptable salt or solvate thereof, in a concentration of approximately 20-40 mg / ml (ii) The buffer solution of approximately 20 mM, (iii) The stabilizer in an amount of approximately 60 to 90 mg / ml, (iv) A pharmaceutical preparation according to any one of claims 1 to 12, comprising the surfactant in an amount of about 0.1 to 0.3 mg / ml.

14. The buffer solution is either a combination of histidine and histidine hydrochloride, or histidine. The stabilizer is sucrose, trehalose, or a combination of sucrose and mannitol, and / or The pharmaceutical formulation according to any one of claims 1 to 13, wherein the surfactant is polysorbate 80.

15. The aforementioned pharmaceutical preparation is (i) an antibody-drug conjugate targeting Claudin 18.2, or a pharmaceutically acceptable salt or solvate thereof, in a concentration of approximately 20-40 mg / ml. (ii) The buffer solution, which is approximately 20 mM, and is a combination of histidine and histidine hydrochloride, or histidine, (iii) The stabilizer in an amount of approximately 60 to 90 mg / ml, wherein the stabilizer is sucrose, trehalose, or a combination of sucrose and mannitol, and (iv) Contains approximately 0.1 to 0.3 mg / ml of polysorbate 80, The pharmaceutical formulation according to any one of claims 1 to 14, wherein the pH of the formulation is about 6.0 to 6.5, preferably about 6.

5.

16. The aforementioned pharmaceutical preparation is (i) Approximately 20 mg / ml of the antibody-drug conjugate targeting Claudin 18.2 or a pharmaceutically acceptable salt or solvate thereof, (ii) The buffer solution, which is approximately 20 mM, and is a combination of histidine and histidine hydrochloride, or histidine, (iii) The stabilizer in an amount of approximately 8% (w / v), which is sucrose, trehalose, or a combination of sucrose and mannitol, and (iv) Contains approximately 0.02% (w / v) of polysorbate 80, The pharmaceutical preparation according to any one of claims 1 to 14, wherein the pH of the preparation is approximately 6.

5.

17. A lyophilized pharmaceutical preparation comprising an antibody-drug conjugate, wherein after reconstitution, it forms the pharmaceutical preparation according to any one of claims 1 to 16.

18. The lyophilized pharmaceutical preparation is a lyophilized preparation comprising the antibody-drug conjugate according to claim 17, which is prepared by lyophilizing the pharmaceutical preparation according to any one of claims 1 to 17.

19. A lyophilized pharmaceutical preparation containing an antibody-drug conjugate, (i) an antibody-drug conjugate in an amount of approximately 1% to 60% (w / w), preferably approximately 15% to 40% (w / w), or a pharmaceutically acceptable salt or solvate thereof. (ii) A buffer solution of approximately 0.5% to 10% (w / w), preferably approximately 2% to 5% (w / w), (iii) Approximately 20% to 90% (w / w), preferably about 50% to 85% (w / w) of a stabilizer, and / or (iv) comprising about 0.05% to 2% (w / w), preferably about 0.1% to 0.5% (w / w), a surfactant, The lyophilized pharmaceutical preparation, wherein the antibody-drug conjugate, the buffer, the stabilizer and / or the surfactant are as defined in claims 1 to 16.

20. A liquid pharmaceutical formulation comprising an antibody-drug conjugate, wherein the liquid pharmaceutical formulation comprising the antibody-drug conjugate is obtained by reconstituting the lyophilized pharmaceutical formulation according to claims 17 to 19 with a vehicle having a pH of about 5.5 to 7.5, preferably about 6.0 to 6.5, more preferably 6.

5.

21. The liquid pharmaceutical preparation comprises the antibody-drug conjugate described in claim 20, which is used for injection or infusion administration, such as intravenous injection, subcutaneous injection, or intramuscular injection.

22. A delivery device comprising a pharmaceutical formulation according to any one of claims 1 to 16, or a lyophilized pharmaceutical formulation comprising an antibody-drug conjugate according to any one of claims 17 to 19, or a liquid pharmaceutical formulation comprising an antibody-drug conjugate according to claim 20 or 21.

23. A pre-filled syringe for use by intravenous, subcutaneous, or intramuscular injection, comprising a pharmaceutical formulation according to any one of claims 1 to 16, a lyophilized pharmaceutical formulation comprising an antibody-drug conjugate according to any one of claims 17 to 19, or a liquid pharmaceutical formulation comprising an antibody-drug conjugate according to claim 20 or 21.

24. Use in the manufacture of a pharmaceutical preparation according to any one of claims 1 to 16, or a lyophilized pharmaceutical preparation comprising an antibody-drug conjugate according to any one of claims 17 to 19, or a liquid pharmaceutical preparation comprising an antibody-drug conjugate according to claim 20 or 21, wherein the pharmaceutical preparation is used to treat or prevent a tumor. More preferably, the tumor is cancer, such as epithelial carcinoma or gastrointestinal cancer, such as gastric cancer, gastroesophageal junction cancer, pancreatic cancer, colorectal cancer or colon cancer, as described above.

25. A pharmaceutical formulation according to any one of claims 1 to 16, or a lyophilized pharmaceutical formulation comprising an antibody-drug conjugate according to any one of claims 17 to 19, or a liquid pharmaceutical formulation comprising an antibody-drug conjugate according to claim 20 or 21, and a pharmaceutical combination comprising one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

26. A kit of parts comprising a pharmaceutical formulation according to any one of claims 1 to 16, or a lyophilized pharmaceutical formulation comprising an antibody-drug conjugate according to any one of claims 17 to 19, or a liquid pharmaceutical formulation comprising an antibody-drug conjugate according to claim 20 or 21, or a pharmaceutical combination according to claim 25, wherein the kit of parts is in the form of a drug administration unit.

Images