Formulations comprising antibody-drug conjugates targeting Claudin18.2, methods of making and uses thereof

Abstract

The present invention relates to a pharmaceutical formulation comprising an antibody-drug conjugate targeting Claudin18.2, to a method for preparing said pharmaceutical formulation, and to the therapeutic and / or prophylactic use of said pharmaceutical formulation.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations of antibody-drug conjugates (ADCs) targeting Claudin18.2 (CLDN18.2). More specifically, the present invention relates to pharmaceutical preparations comprising antibody-drug conjugates targeting Claudin18.2, especially lyophilized preparations, liquid preparations and reconstituted liquid preparations, methods for preparing the pharmaceutical preparations, and therapeutic and / or preventive uses of the pharmaceutical preparations. Background of the Invention

[0003] Claudins are a family of proteins that are important components of tight junctions. They establish intercellular barriers that control the flow of molecules between cells. Claudins family proteins have four transmembrane domains, both the N-terminus and the C-terminus are included in the cytoplasm. Different Claudins proteins are expressed on different tissues, and changes in their functions are related to the formation of cancer in various tissues. For example, Claudin-1 is expressed in colon cancer and has prognostic value, Claudin-18 is highly expressed in gastric cancer 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.

[0004] Claudin-18 isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker, and its expression in normal tissues is strictly limited to differentiated epithelial cells of the gastric mucosa, but not expressed in the gastric stem cell region. CLDN18.2 is expressed in a considerable portion of primary gastric cancers, and its expression level is retained in gastric metastatic cancer tissues. In addition to gastric cancer, CLDN18.2 expression has also been found in pancreatic cancer, making it 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 ).

[0005] Regarding gastric cancer, in 2014, there were approximately 410,000 new cases of gastric cancer and approximately 290,000 deaths in China, accounting for almost half of the global cases and deaths, and the number is still increasing. However, there are a large number of unmet clinical tumor treatment needs, so it is necessary to develop drugs targeting Claudin18.2.

[0006] Despite the clinical success of therapeutic antibodies, naked MAbs targeting cell surface tumor antigens rarely provide sufficient efficacy alone. To increase the low activity of MAbs, new strategies focus on binding them to toxic molecules. Plant and bacterial toxins and small chemotherapeutic molecules can be good candidates because they are very effective and active in very small amounts.

[0007] Due to technological advances made during the past few years, the field of antibody-drug conjugates (ADCs) for the treatment of cancer has recently experienced an increasing development activity by pharmaceutical companies aimed at solving the problems they initially presented with regard to immunogenicity, affinity, specificity, undesirable toxicity, yield, half-life, etc.

[0008] Despite some progress, there is still a need for other therapeutic strategies for treating tumors, and for components used in such therapeutic strategies, in particular, there is a need for anti-Claudin18.2 antibodies with high affinity, good specificity and / or low immunogenicity risk, and for ADC molecules with higher activity, low toxicity, long half-life, good specificity or affinity, and / or good half-life or drugability.

[0009] There is a need for new pharmaceutical formulations containing antibody-drug conjugates targeting Claudin18.2 that are sufficiently stable and suitable for administration. The pharmaceutical formulations, especially lyophilized formulations, have good stability and remain stable during storage and transportation, and the lyophilized formulations are easy to administer after reconstitution. SUMMARY OF THE INVENTION

[0011] The present invention provides a pharmaceutical preparation comprising an antibody-drug conjugate (ADC) targeting Claudin18.2.

[0012] 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; wherein the pH of the formulation is about 5.5-7.5, for example, about 6.0-6.5, for example 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.

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

[0014]

[0015]

[0016] Wherein Ab is an antibody or an antigen-binding fragment thereof targeting Claudin18.2,

[0017] Where q represents the average DAR,

[0018] In IEX019-02, IEX019-04, and IEX019-05, q is 2-5, for example, 3-5 or 3.5-4.5 or 3-4, and in IEX019-03, q is 5-11, for example, 7-9 or 7.5-8.5.

[0019] According to the context, it should be understood that when the variable after the square brackets of the ADC structural formula is indicated as the average DAR, such as q in the above structural formula, it represents the ratio of the drug part (e.g., Exatecan) to the Ab determined by a conventional determination method, as described in the definition section herein. The average DAR can be a decimal. For the average DAR, for example, when "q is 3-5" or "q is a numerical value of 3-5" is mentioned, it means that q is any number selected from 3 to 5, including integers and decimals.

[0020] In some embodiments, the antibody-drug conjugate targeting Claudin18.2 is selected from:

[0021]

[0022]

[0023]

[0024] Wherein Ab is an antibody or an antigen-binding fragment thereof targeting Claudin18.2,

[0025] In Formula IA, y is an integer of 1 or 2, and in Formula IB, IC or ID, y is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0026] It should be understood that when the variable after the square brackets in the ADC structural formula is not specified as the average DAR, such as y in Formulas IA to ID, it does not represent the average DAR, but refers to the number of Linker-payloads (i.e., the structures in the square brackets of Formulas IA to ID) connected to each Ab in the ADC molecule, unless otherwise specified or contradictory according to the context. For example, when y is 2, it means that 1 Ab is connected to 2 Linker-payloads in the ADC molecule.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof targeting Claudin18.2 comprises HCDR1, HCDR2 and HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7 and 8, respectively.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof targeting Claudin18.2 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region

[0029] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 4; or

[0030] (ii) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acids of SEQ ID NO:4; or

[0031] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, preferably, the amino acid changes do not occur in the CDR regions; and / or

[0032] Light chain variable region

[0033] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or

[0034] (ii) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acids of SEQ ID NO:9; or

[0035] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 9, preferably, the amino acid changes do not occur in the CDR regions.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof targeting Claudin18.2 comprises a heavy chain and a light chain, wherein

[0037] Heavy chain

[0038] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 11; or

[0039] (ii) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acids of SEQ ID NO: 11; or

[0040] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 11; and / or

[0041] Light chain

[0042] (i) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or

[0043] (ii) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acids of SEQ ID NO: 12; or

[0044] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 12.

[0045] In some embodiments, the buffer is selected from one or more of histidine, citric acid, succinic acid, glutamic acid, phosphoric acid, acetic acid or salts thereof; preferably, the buffer is a combination of histidine and histidine hydrochloride or histidine.

[0046] In some embodiments, the stabilizer is selected from one or more of sugars and polyols; preferably, the sugars are selected from sucrose, trehalose, glucose, lactose, maltose, cyclodextrin, maltodextrin and dextran, and the polyols are selected from mannitol, sorbitol and xylitol; more preferably, the stabilizer is selected from sucrose, trehalose, mannitol or a combination thereof.

[0047] In some embodiments, the surfactant is selected from alkyl poly(ethylene oxide), polysorbate, pluronic or a combination thereof; preferably, selected from polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40.

[0048] In some embodiments, the buffer is a combination of histidine and histidine hydrochloride or is histidine;

[0049] The stabilizer is sucrose; trehalose; or a combination of sucrose and mannitol; and / or

[0050] The surfactant is polysorbate 80.

[0051] In some embodiments, the pharmaceutical preparation contains about 1-200 mg / ml, preferably 15-60 mg / ml, more preferably about 20-40 mg / mL of an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof.

[0052] In some embodiments, the pharmaceutical formulation further comprises about 1-100 mM, preferably about 5-50 mM, more preferably about 10-30 mM of a buffer.

[0053] In some embodiments, the pharmaceutical formulation further comprises about 10-200 mg / ml, preferably about 50-100 mg / ml, more preferably about 60-90 mg / ml of a stabilizer.

[0054] In some embodiments, the pharmaceutical formulation further comprises about 0.01-5 mg / ml, preferably about 0.05-1 mg / ml, more preferably about 0.1-0.3 mg / ml of a surfactant.

[0055] In some embodiments, the pharmaceutical formulation comprises:

[0056] (i) about 20-40 mg / ml of an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof;

[0057] (ii) about 20 mM buffer;

[0058] (iii) about 60-90 mg / ml of a stabilizer; and

[0059] (iv) about 0.1-0.3 mg / ml surfactant.

[0060] In some embodiments, the pharmaceutical formulation comprises:

[0061] (i) about 20-40 mg / ml of an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof;

[0062] (ii) about 20 mM buffer, the buffer being a combination of histidine and histidine hydrochloride or histidine;

[0063] (iii) about 60-90 mg / ml of a stabilizer, said stabilizer being sucrose; trehalose; or a combination of sucrose and mannitol; and

[0064] (iv) about 0.1-0.3 mg / ml polysorbate 80;

[0065] The pH of the liquid preparation is about 6.0-6.5, preferably about 6.5.

[0066] In some embodiments, the pharmaceutical formulation comprises:

[0067] (i) about 20 mg / ml of an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof;

[0068] (ii) about 20 mM buffer; the buffer is a combination of histidine and histidine hydrochloride or histidine;

[0069] (iii) about 8% (w / v) stabilizer; the stabilizer is sucrose; trehalose; or a combination of sucrose and mannitol; and

[0070] (iv) about 0.02% (w / v) polysorbate 80;

[0071] wherein the pH of the formulation is about 6.5.

[0072] In a second aspect, the present invention provides a lyophilized pharmaceutical preparation comprising an antibody-drug conjugate, wherein the lyophilized pharmaceutical preparation forms or is capable of providing the pharmaceutical preparation as described in the first aspect above after reconstitution.

[0073] In some embodiments, the lyophilized pharmaceutical preparation is prepared by lyophilizing the pharmaceutical preparation of the first aspect.

[0074] In a third aspect, the present invention provides a lyophilized pharmaceutical preparation comprising an antibody-drug conjugate, comprising

[0075] (i) about 1%-60% (w / w), preferably about 15%-40% (w / w), of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof;

[0076] (ii) about 0.5%-10% (w / w), preferably about 2%-5% (w / w), of a buffer;

[0077] (iii) about 20% to 90% (w / w), preferably about 50% to 85% (w / w), of a stabilizer; and / or

[0078] (iv) about 0.05% to 2% (w / w), preferably about 0.1% to 0.5% (w / w), of a surfactant;

[0079] The antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, a buffer, a stabilizer and / or a surfactant are as defined in the first aspect above.

[0080] In a fourth aspect, the present invention provides a liquid pharmaceutical preparation containing an antibody-drug conjugate, wherein the liquid pharmaceutical preparation containing an antibody-drug conjugate is obtained by reconstituting the above-mentioned lyophilized pharmaceutical preparation with a solvent, and has a pH of about 5.5-7.5, preferably about 6.0-6.5, and more preferably about 6.5.

[0081] In some embodiments, the liquid pharmaceutical formulation is for administration by injection or infusion, such as for intravenous injection, subcutaneous injection, or intramuscular injection.

[0082] In a fifth aspect, the present invention provides a delivery device comprising the pharmaceutical formulation, lyophilized pharmaceutical formulation or liquid pharmaceutical formulation of the present invention.

[0083] In a sixth aspect, the present invention provides a prefilled syringe comprising the pharmaceutical formulation, lyophilized pharmaceutical formulation or liquid pharmaceutical formulation of the present invention for intravenous injection, intramuscular injection or subcutaneous injection.

[0084] In a seventh aspect, the present invention provides the use of the pharmaceutical preparation, lyophilized pharmaceutical preparation and liquid pharmaceutical preparation of the present invention in preparing a drug. Preferably, the pharmaceutical preparation, lyophilized pharmaceutical preparation and liquid pharmaceutical preparation are used to prevent or treat tumors.

[0085] More preferably, the tumor is a cancer, such as an epithelial cancer or a cancer of the digestive tract, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.

[0086] In an eighth aspect, the present invention provides a pharmaceutical combination,

[0087] The drug set comprises the drug formulation, lyophilized drug formulation, or liquid drug formulation of the present invention and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

[0088] In a ninth aspect, the present invention provides a kit of parts, comprising the pharmaceutical preparation, lyophilized pharmaceutical preparation, or liquid pharmaceutical preparation or the pharmaceutical combination of the present invention, preferably the kit is in the form of a pharmaceutical dosage unit.

[0089] The preparation of the antibody-drug conjugate targeting Claudin18.2 of the present invention has various advantages and / or benefits:

[0090] (1) The preparation of the present invention exhibits excellent stability to various factors affecting stability (such as temperature, vibration, freeze-thaw, and light). After long-term storage or accelerated experiments, the appearance, protein concentration, insoluble particles, DAR, etc. of the preparation do not change significantly, and the antibody activity is maintained well, which is conducive to long-term storage or transportation;

[0091] (2) The lyophilized preparation of the present invention is rapidly reconstituted, and the physicochemical properties and activity of the antibody-drug conjugate do not change significantly after reconstitution, which is convenient for patient administration;

[0092] (3) The preparation process of the preparation of the present invention is simple and has high repeatability.

[0093] In addition, the conjugate in the preparation of the present invention has the following advantages:

[0094] (1) Bind to target cells expressing human CLDN18.2 with high affinity;

[0095] (2) Ability to enter cells through endocytosis and kill target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency;

[0096] (3) There is a significant bystander effect;

[0097] (4) High anti-tumor efficacy;

[0098] (5) Low toxicity;

[0099] (6) Good stability;

[0100] (7) It has good drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 The cell binding effect of the IEX019 molecule is shown.

[0102] Figure 2 The endocytosis of IEX019 molecules on DANG-hCLDN18.2 cells is shown.

[0103] Figure 3 The killing effect of IEX019 molecule on cell lines with low expression of hCLDN18.2 was shown ( Figure 3A) The killing effect of IEX019 on hCLDNA18.2 medium-expression cell lines ( Figure 3 B) and the killing effect of IEX019 molecules on cell lines with high expression of hCLDN18.2 ( Figure 3 C).

[0104] Figure 4 The bystander effect of the IEX019 molecule is shown.

[0105] Figure 5 IEX019 showed tumor suppressive efficacy in mice ( Figure 5 A) and weight changes ( Figure 5 B).

[0106] Figure 6 IEX019 showed tumor suppressive efficacy in mice ( Figure 6 A) and weight changes ( Figure 6 B).

[0107] Figure 7 IEX019 showed tumor suppressive efficacy in mice ( Figure 7 A) and weight changes ( Figure 7 B).

[0108] Figure 8 DSC spectra for ADC pH buffer screening are shown. DETAILED DESCRIPTION OF THE INVENTION

[0110] I. Definitions

[0111] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs.

[0112] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0113] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0114] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0115] As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the elements, integers or steps described. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0116] The term "CLAUDIN" or "CLDN" used in this article is the most important skeletal protein that determines the tight junction structure between cells. It participates in adhesion junctions and plays an important role in the metastasis and invasion of tumor cells. Claudin protein is widely present in mammalian epithelial and endothelial cells, and its distribution is mainly on the side of epithelial cells and on the plasma membrane of basal cells. Different Claudin proteins have their own specific expression in different tissues. Among them, the Claudin18 (CLDN18) gene is located at 3q22.3, with a molecular weight of 24kDa and 261 amino acid residues. It is a member of the Claudins superfamily, and its protein structure includes 2 extracellular loops and 4 transmembrane regions. The two subtypes of 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). In the primary structure sequence of the two proteins, only the amino acid residues at certain positions from the N-terminal signal peptide to the extracellular loop 1 (Loop1) structure differ, especially in the extracellular loop 1, CLDN18.1 and CLDN18.2 only have 8 amino acids different. The sequence homology between species of the two subtypes of CLDN18 is also very high. Among them, the extracellular loop 1 of CLDN18.2 is completely consistent in sequence in different species such as humans, mice, and macaques, and the homology of CLDN18.2 proteins between humans and mice reaches 84%, indicating that the CLDN18.2 protein sequence is extremely conservative (O. Tureci et al., Gene 481: 83-92, 2011). CLDN18.2 or any variants and isoforms thereof can be isolated from cells or tissues that naturally express them, or recombinantly produced using techniques well known in the art and / or those described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0117] The terms "anti-CLDN18.2 antibody", "anti-CLDN18.2", "CLDN18.2 antibody", or "antibody that binds to CLDN18.2", or "antibody that specifically binds to CLDN18.2", or "antibody that targets CLDN18.2" as used herein refer to an antibody that is capable of binding to (human) CLDN18.2 with sufficient affinity so that the antibody can be used as a therapeutic agent 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 without binding to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), MSD electrochemiluminescence detection technology assay or surface plasmon resonance (SPR) or flow cytometry. The expression of CLDN18.2 in cells can be determined in a variety of ways, such as anti-CLDN18.2 antibodies. For example, the binding strength of "highly expressed CLDN18.2" cells to anti-CLDN18.2 antibodies (e.g., determined by FACS) can be 500, 600, 700, 800, 900 or preferably 1000 times greater than the binding strength of anti-CLDN18.2 antibodies to cells that do not express CLDN18.2, such as 1100 times, 1200, 1300, 1400 times or more. For example, cells that "moderately express CLDN18.2" may bind to an anti-CLDN18.2 antibody with an intensity (e.g., as determined by FACS) that is 5-500 times, such as 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 times or more, but not more than 500 times, as strongly as the anti-CLDN18.2 antibody binds to cells that do not express CLDN18.2.

[0118] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules having the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, full-length antibodies comprise two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, full-length antibodies comprise two heavy chains (H) interconnected by disulfide bonds.

[0119] For conventional four-chain IgG antibodies, the full-length antibody heavy chain is usually composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0120] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0121] "Antigen-binding fragment" refers to a molecule that is different from an intact antibody, comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibody; single-chain antibody (e.g., scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.

[0122] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. The skilled person will understand that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant 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.

[0123] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (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)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0124] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0125]

[0126] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0127] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

[0128] Unless otherwise indicated, in the present invention, when referring to the residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position 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)).

[0129] In one embodiment, the heavy chain variable region CDR of the antibody of the present invention is determined according to the following rules:

[0130] VH CDR1 was determined according to the AbM rule; and VH CDR2 and CDR3 were both determined according to the Kabat rule.

[0131] In one embodiment, the light chain variable region CDRs of the antibodies of the invention are determined according to the Kabat rules.

[0132] In one embodiment, the heavy chain variable region CDRs of the antibody of the invention are determined according to the following rules: VH CDR1 is determined according to the AbM rule; and VH CDR2 and CDR3 are both determined according to the Kabat rule; and each CDR of the light chain variable region is determined according to the Kabat rule.

[0133] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0134] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs are different from antibody to antibody, only a limited number of amino acid positions in the CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As those skilled in the art will appreciate, the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0135] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. A natural immunoglobulin "Fc domain" comprises two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a natural antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or comprises the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Herein, the term "Fc region" does not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region CL of an immunoglobulin, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases.

[0136] "Antibody of IgG form" is used to define the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant region differs between different types of antibodies. For example, an antibody of IgG4 form means that its heavy chain constant region is from IgG4, or an antibody of IgG1 form means that its heavy chain constant region is from IgG1.

[0137] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0138] As used herein, "antibody-drug conjugate (ADC)" refers to a structure / compound obtained by linking an antibody to a drug, typically via a linker.

[0139] The general term "sugar" used herein indicates monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" used herein means a derivative of a monosaccharide, i.e. a monosaccharide comprising a substituent and / or a functional group. Examples of sugar derivatives include amino sugars and sugar 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 sugar derivatives also include compounds represented herein as E(A)x, wherein E is a sugar or a sugar derivative, and wherein E includes x functional groups A.

[0140] The core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as a GlcNAc bonded to the antibody via C1, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. The core-GlcNAc substituent may be present at the native glycosylation site of the antibody, but it may also be introduced at a different site of the antibody. Herein, the core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fucα1-6)GlcNAc substituent, also referred to as GlcNAc(Fuc).

[0141] "Glycosylation modification" refers to the process of changing the sugar chains of antibodies by glycosylation engineering. The glycosylation of antibodies can be further modified for various purposes to obtain antibodies with new glycosylation, for example, to remove glycosylation in order to eliminate FcγR affinity and complement binding / effector function, to reduce fucose and sialic acid groups and increase bisecting N-acetylglucosamine, galactose and mannose in order to enhance Fc-mediated ADCC and CDC effects. Glycosylation modification methods are known in the art, for example, by changing the glycosylation sites of antibodies to increase or decrease the sugar chains on the surface of antibodies, or by modifying sugar chains in vitro by chemical or enzymatic methods, or by changing the glycosylation pathway of the expression system (for example, composed of enzymes such as glycosidases and glycosyltransferases) to catalyze the glycosylation of antibodies, and the glycosylation of antibodies can also be changed by the influence of cell culture conditions. In some embodiments, the "glycosylation modification" of the present invention is carried out by modifying sugar chains by enzymatic methods in vitro. Preferably, the glycosylation modification of the present invention is performed by modifying the sugar chain using a glycosidase (eg, endoglycosidase or glycosyltransferase).

[0142] The antibody with modified glycosylation of the present invention refers to an antibody whose glycosylation pattern is modified compared to an antibody with a natural glycosylation pattern. Preferably, the antibody with modified glycosylation refers to an antibody obtained after the antibody is expressed in an expression system (e.g., a mammalian cell) and the sugar chain is modified by an enzymatic method in vitro (e.g., by a glycosidase (e.g., an endoglycosidase or a glycosyltransferase)). More preferably, the antibody with modified glycosylation of the present invention refers to an antibody comprising a core-GlcNAc and a sugar derivative E(A)x connected thereto, wherein the GlcNAc is bonded to the antibody via C1, preferably via an N-glycosidic bond to an amide nitrogen atom on the side chain of an asparagine amino acid of the antibody. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucosylated, the fucose is usually connected to C6 of the -GlcNAc substituent via α-1,6. The fucosylated-GlcNAc substituent refers to core-GlcNAc(Fuc) and the fucosylated GlcNAc-E(A)x substituent refers to GlcNAc(Fuc)-E(A)x.

[0143] The term "site-specific conjugation" as described herein refers to conjugation in which a drug / active substance is specifically linked to a specific site of an antibody via a linker.

[0144] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl group preferably contains 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. Representative 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, n-decyl, etc.

[0145] The term "aryl" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 20, for example 6 to 12 carbon atoms in the ring portion. Preferably, aryl is (C6-C 10 ) aryl. Non-limiting examples include phenyl, biphenyl, naphthyl or tetrahydronaphthyl, each of which may be optionally substituted with 1-4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, thiol, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-OC(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, heterocyclyl, etc., wherein R is independently hydrogen, alkyl, aryl, heteroaryl, aryl-alkyl-, heteroaryl-alkyl-, etc.

[0146] The term "cycloalkyl" is a cyclic alkyl group, that is to say, a monovalent, saturated or unsaturated hydrocarbon group with a ring structure. Cycloalkyl includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups with a ring structure. The cycloalkyl group can contain 3 or more, for example 3-18, 3-10 or 3-8 carbon atoms in the ring, and usually according to the present invention, contains 3 to 6 atoms. The example of the cycloalkyl group includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0147] As used herein, the term "heteroaryl" refers to a 5-20 membered (e.g., 5-14, 5-8, 5-6) monocyclic- or bicyclic- or fused polycyclic ring system containing 1-8 heteroatoms selected from N, O or S. Preferably, heteroaryl is a 5-10 membered ring system. Representative heteroaryl groups include 2- or 3-thienyl, 2- or 3-furanyl, 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- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3- or 4-pyridinyl, 3- or 4-pyridazinyl, 3-, 4- or 5-pyrazinyl, 2-pyrazinyl, 2-, 4- or 5-pyrimidinyl.

[0148] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugate of the present invention, and the salt is not biologically or otherwise undesirable. The ADC conjugate of the present invention may exist in the form of their 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 by the ADC conjugate of the present invention and an organic or inorganic acid, and the organic or inorganic acid 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, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention and organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as ammonium salts formed by organic bases containing N groups.

[0149] The term "solvate" refers to an association formed by one or more solvent molecules and the ADC conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0150] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0151] The term "drug: antibody ratio" or "DAR" refers to the ratio of the small molecule drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety. In some embodiments described herein, for example, the DAR can be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the overall ratio of the small molecule drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in the product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), and this DAR is referred to as the average DAR herein. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 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, 4.4. 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or a range having two of these values ​​as endpoints.

[0152] The term "therapeutic agent" as used herein 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).

[0153] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. In this context, it is sometimes referred to as a "toxin".

[0154] "Free drug" refers to the free form of the anti-tumor compound or cytotoxic agent dissociated from the ADC molecule of the present invention. Free drug is sometimes also referred to herein as free toxin.

[0155] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0156] The term "small molecule drug" refers to low molecular weight organic compounds that can regulate biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD, and preferably less than 10 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 mimics, and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0157] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. An immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0158] The term "effective amount" refers to an amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses administered to a patient, produces the desired effect in a patient in need of treatment or prevention.

[0159] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0160] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0161] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as in the initially transformed cell are included herein.

[0162] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.

[0163] "Individuals" or "subjects" include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0164] An "isolated" antibody or other molecule (e.g., ADC molecule) is an antibody or molecule that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody or ADC molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0165] The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0166] The terms "tumor" and "cancer" are sometimes used interchangeably herein to encompass both solid tumors and hematological tumors.

[0167] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which cell growth is typically unregulated. In certain embodiments, cancers suitable for treatment by the antibody-drug conjugates of the invention include gastric cancer, pancreatic cancer, or gastroesophageal junction cancer, including metastatic forms of those cancers.

[0168] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0169] The term "pharmaceutical excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., which is administered together with the active substance. See "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Seskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0170] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0171] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the ADC molecules in the pharmaceutical preparation of the present invention, and (ii) other therapeutic agents) are administered to the patient in sequence as separate entities, without specific time restrictions or at the same or different time intervals, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient. In some embodiments, the ADC molecules of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those when they are used alone. The term "fixed combination" means that two or more active agents are administered to the patient simultaneously in the form of a single entity. It is preferred that the dosage and / or time interval of the two or more active agents be selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate preparation, which can be the same or different.

[0172] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to a desired dose before administration. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating a disorder or condition described herein.

[0173] As used herein, "treat," ...

[0174] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0175] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0176] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0177] The term "lyophilized preparation" refers to a composition obtained by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0178] The term "buffer" refers to a pH buffer that can keep the pH value of a solution stable. Preferably, the buffer can keep the pH of the liquid preparation of the present invention or the liquid preparation obtained after reconstitution of the lyophilized preparation of the present invention at about 5.5-7.5, for example, about 6.0-6.5, about 5.5-7.2, about 6.0-7.0, or about 6.3-6.8. Preferably, the buffer is selected from one or more of histidine, citric acid, succinic acid, glutamic acid, phosphoric acid, acetic acid or a salt thereof. Wherein, the salt includes but is not limited to salts formed with acids such as hydrochloric acid or sulfuric acid, salts formed with alkali metals, such as lithium, sodium or potassium salts, such as histidine hydrochloride.

[0179] The term "stabilizer" refers to a chemical substance that can maintain the stability of the formulation at the chemical, physical and / or biological level. Preferably, the stabilizer is selected from one or more of sugars and polyols. The sugars may be selected from, but not limited to, sucrose, trehalose, glucose, lactose, maltose, cyclodextrin, maltodextrin and dextran. The polyols may be selected from, but not limited to, mannitol, sorbitol and xylitol. The sugars or polyols may be in the form of hydrates.

[0180] 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 liquid preparation is, for example, about 0.01-5 mg / ml, such as 0.01-2, about 0.05-1, about 0.05-2, about 0.1-0.5, about 0.1-0.4, about 0.1-0.3, about 0.2-0.4 mg / ml. Preferably, the surfactant is a nonionic surfactant, including but not limited to, alkyl poly (ethylene oxide); polysorbate, such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Pluronic; or a combination thereof.

[0181] The term "reconstituted" formulation refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (eg, a lyophilized formulation) in a physiologically acceptable solution. In this document, in some cases, "reconstitution" and "reconstitution" are used interchangeably for formulations.

[0182] As used herein, "w / v" means "weight / volume", for example, "1% w / v" means 1 g / 100 ml = 0.01 g / ml = 10 mg / ml.

[0183] II. Implementation Plan

[0184] In one 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, wherein the pH of the formulation is about 5.5-7.5, preferably about 6.0-6.5, and more preferably about 6.5.

[0185] In one embodiment, the antibody-drug conjugate targeting Claudin 18.2 is represented by Formula I:

[0186]

[0187] in

[0188] L1 is the connector;

[0189] E is a sugar or a sugar 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);

[0190] And GlcNAc is N-acetylglucosamine, Fuc is fucose;

[0191] D is an anti-tumor drug, preferably Exatecan, DXd, MMAE or DM1;

[0192] r is 1-5, for example 1, 2, 3, 4 or 5, preferably 1 or 2;

[0193] b is 0 or 1;

[0194] where x is 1 or 2; and

[0195] y is 1 to 10; for example 1, 2, 3, 4, 5, 6, 7 or 8;

[0196] Preferably x is 1 and y is 2.

[0197] In some embodiments, E is GalNAc.

[0198] In some embodiments, E is 6-deoxy-2-acetamidogalactose, and it is linked to L1 via the 6-C atom.

[0199] In some embodiments, the GlcNAc attached to the Ab is present at the Asn297-glycosylation site of both heavy chains of the antibody.

[0200] In some embodiments, L1 has the structure

[0201]

[0202] Q is -N(H)C(O)CH2- or -CH2-;

[0203] R1 is independently selected from hydrogen, halogen, -OR2, -NO2, -CN, -S(O)2R2, C1-C 12 Alkyl, aryl, heteroaryl, C1-C 12 Alkyl aryl, C1-C 12 Alkyl heteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 alkyl, and wherein the alkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, arylalkyl and heteroarylalkyl are optionally substituted, wherein two substituents R1 can be linked together to form a fused, bridged or spiro cycloalkyl or a fused or spiro aromatic or heteroaromatic substituent, and wherein R2 is independently selected from hydrogen, halogen, C1-C 24 Alkyl, aryl, heteroaryl, C1-C 12 Alkyl aryl, C1-C 12 Alkyl heteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 alkyl;

[0204] R3 and R4 are each independently selected from hydrogen, halogen, C1-C 24 Alkyl, aryl, heteroaryl, C1-C 12 Alkyl aryl, C1-C 12 Alkyl heteroaryl, aryl C1-C 12 Alkyl and heteroaryl C1-C 12 alkyl;

[0205] c is 0, 1, 2, 3, or 4;

[0206] m is 0 or 1;

[0207] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0208] Y is O, S or NR1;

[0209] L2 and L3 are each independently C1-C 12 An alkyl group in which one or more (e.g., 1, 2, 3 or 4) carbon atoms are optionally replaced by a heteroatom selected from O, N and S, provided that said O, N and / or S are not directly connected to each other;

[0210] Each L4 is independently a cleavable linker.

[0211] In some embodiments, L4 is each independently

[0212] wherein R5 and R6 are each independently selected from hydrogen and C1-C 12 alkyl; and

[0213] Ar is selected from aryl groups, preferably phenyl groups.

[0214] In some embodiments, L4 is each independently

[0215] In some embodiments, -L1- has the structure

[0216]

[0217] In some embodiments, the antibody-drug conjugate targeting claudin18.2 has an average DAR of 1-15, such as 2-10, 2-8 or 3-5.

[0218] In some embodiments, the antibody-drug conjugate targeting claudin18.2 is selected from:

[0219]

[0220]

[0221]

[0222] Wherein Ab is an antibody or antigen-binding fragment thereof targeting Claudin18.2

[0223] Wherein q represents the average DAR. In IEX019-02, IEX019-04, and IEX019-05, q is 2-5, such as 3-5 or 3.5-4.5. In IEX019-03, q is 5-11, such as 7-9 or 7.5-8.5.

[0224] In some embodiments, the heavy chain constant region HC of the present invention is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1, such as a wild-type IgG1 heavy chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a lambda or kappa light chain constant region.

[0225] In some specific embodiments of the present invention, the Claudin18.2 antibody of the present invention comprises HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, and / or LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7 and 8, respectively.

[0226] In some embodiments of the present invention, the Claudin18.2 antibody of the present invention comprises VH and / or VL, wherein the amino acid sequence of VH is shown by SEQ ID NO:4, and the amino acid sequence of VL is shown by SEQ ID NO:9.

[0227] 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 amino acid sequence of the heavy chain is shown by SEQ ID NO:11, and the amino acid sequence of the light chain is shown by SEQ ID NO:12.

[0228] In one embodiment of the invention, the amino acid changes described herein include replacement, insertion or deletion of amino acids. Preferably, the amino acid changes described herein are amino acid replacements, preferably conservative replacements. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid changes described herein occur in the Fc region of the heavy chain constant region of an antibody.

[0229] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to an amino acid substitution by another amino acid within the same class, such as an acidic amino acid substitution by another acidic amino acid substitution, a basic amino acid substitution by another basic amino acid substitution, or a neutral amino acid substitution by another neutral amino acid substitution. In certain embodiments, the substitution occurs in the CDR region of an antibody. Typically, the variant obtained has modifications (e.g., improvements) and / or will have certain biological properties that are substantially retained relative to the parent antibody in terms of certain biological properties (e.g., increased affinity). An exemplary substitution variant is an affinity matured antibody.

[0230] The Claudin18.2 antibody of the present invention may be an antibody with modified glycosylation. In some embodiments, the antibody is an antibody obtained after enzymatic modification of sugar chains in vitro (e.g., modification of sugar chains by glycosidases (e.g., endoglycosidases or glycosyltransferases)). In some embodiments, the antibody with modified glycosylation refers to an antibody in which the sugar chains on the glycosylation sites of the antibody are modified from non-uniformly structured N sugar chains to single-structured N sugar chains with reactive groups (e.g., any reactive groups that can react with the linker moiety, such as azide, keto, and alkyne). In a preferred embodiment, the N-glycosylation site of the antibody is a conserved N-glycosylation site on the Fc domain of the antibody, such as Asn297.

[0231] Methods for modifying antibody glycosylation suitable for the present invention are described, for example, in PCT / NL2013 / 050744, PCT / EP2016 / 059194 or PCT / EP2017 / 052792, which are incorporated herein in their entirety.

[0232] In a preferred embodiment, the GlcNAc-E(A)x substituent is present at the Asn297-glycosylation site of both heavy chains of the antibody.

[0233] In some embodiments, L in formula (I) of the present invention is a linker. Any linker known in the art can be used to connect to the anti-human CLDN18.2 of the present invention, preferably, the linker can achieve site-specific coupling of ADC.

[0234] In some embodiments, the linker suitable for the present invention can be any linker capable of achieving conjugation of an antibody to a drug. In some embodiments, the linker can be a linker used in a technique capable of achieving site-specific conjugation.

[0235] In a preferred embodiment, the linker of the present invention is a linker connected to an antibody oligosaccharide. The "linker connected to an antibody oligosaccharide" defined herein refers to any linker that is connected to a reactive group on a sugar chain at an antibody glycosylation site to couple the antibody to a drug. The sugar chain at the antibody glycosylation site is generally an N-sugar chain, and is usually modified to transform the non-uniform structure N-sugar chain into a single structure N-sugar chain with a reactive group, and then the reactive group carried by the sugar chain is connected to the "linker" to achieve site-specific coupling of the drug to the antibody to obtain an antibody-drug conjugate.

[0236] In one embodiment, the "linker attached to an antibody oligosaccharide" of the present invention is particularly any linker that can achieve site-specific coupling with a reactive group on the N-sugar chain at a conserved N-glycosylation site (e.g., Asn297) on the antibody Fc domain, such as the linker described in PCT / NL2013 / 050744 or the linker of PCT / EP2021 / 075401, which are incorporated herein in their entirety.

[0237] In one embodiment, the reactive group of the present invention is an azido group, a keto group or an alkynyl group. In one embodiment, the linker of the present invention is a linker comprising an alkynyl group. In one embodiment, the reactive group of the present invention is an azido group, a keto group or an alkynyl group, preferably an azido group, and the linker of the present invention is a linker comprising an alkynyl group. When the present invention refers to such linkers, since the reactive group reacts with the group of the linker to form a new group, the group formed after the reactive group in the antibody-drug conjugate reacts can also be defined as a part of the "linker", for example, as shown in formula (I) of the present invention.

[0238] Linkers suitable for use in the present invention also 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 or MD linkers; acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, and ultraviolet-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 linker applicable to the present invention can also be a combination of one or more linkers, for example, a linker degraded by cathepsin can be combined with other types of linkers to form a new linker. Therefore, the "linker" described in the present invention covers a single type of linker, or a combination of different types of linkers, as long as it can couple the antibody of the present invention to the drug. Therefore, in one embodiment, the linker applicable to the present invention is MC-VC-PAB, vc-PAB, SMCC or MC-GGFG.

[0239] D in formula (I) of the present invention can be any antitumor compound, as long as it has an antitumor effect and has a substituent or a partial structure that can be connected to the linker structure, without any particular limitation. For example, the antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For antitumor compounds, part or all of the linker can preferably be cut off in tumor cells to free the antitumor compound part, thereby showing an antitumor effect. When the linker is cut off from the connecting part of the drug, the antitumor compound is freed with an unmodified structure, and its original antitumor effect can be exerted.

[0240] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent or a chemotherapeutic agent, such as a camptothecin compound Exatecan (topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), an auristatin compound such as monomethyl auristatin E (MMAE) or a maytansine compound such as a small molecule microtubule inhibitor DM1. The structures in the examples of the present application show the structures of representative compounds of these anti-tumor compounds.

[0241] In some embodiments, the buffer is selected from one or more of histidine, citric acid, succinic acid, glutamic acid, phosphoric acid, acetic acid or their salts. Wherein, the salt of amino acid (histidine, glutamic acid) includes but is not limited to the salt formed by the amino acid with hydrochloric acid or sulfuric acid. The salts of phosphoric acid, acetic acid, succinic acid, citric acid include but are not limited to alkali metal salts of the corresponding acid such as lithium, sodium or potassium salts. Preferably, the histidine salt is histidine hydrochloride. Preferably, the buffer is selected from a combination of histidine and histidine hydrochloride, and histidine.

[0242] In some embodiments, the stabilizer is selected from one or more of sugars and polyols, preferably, the sugars are selected from but not limited to: sucrose, trehalose, glucose, lactose, maltose, cyclodextrin, maltodextrin and dextran, and the polyols are selected from but not limited to: mannitol, sorbitol and xylitol. The sugars or polyols may be in the form of hydrates.

[0243] 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.

[0244] In some embodiments,

[0245] The buffer is a combination of histidine and histidine hydrochloride or histidine;

[0246] The stabilizer is sucrose; trehalose; or a combination of sucrose and mannitol; and / or

[0247] The surfactant is polysorbate 80.

[0248] In some embodiments, the concentration of the antibody-drug conjugate targeting claudin18.2 or a pharmaceutically acceptable salt or solvate thereof in the pharmaceutical preparation is about 1-200 mg / ml, for example, about 5-100 mg / ml, about 10-80 mg / ml, about 15-60 mg / ml, about 20-50 mg / ml, about 20-40 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 15-60 mg / ml, more preferably about 20-40 mg / mL.

[0249] In some embodiments, the concentration of the buffer in the pharmaceutical formulation is about 1-100 mM, about 5-50 mM, about 5-30 mM, about 5-20 mM or about 10-30 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 5-50 mM, more preferably about 10-30 mM.

[0250] 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. When the stabilizer is a plurality of components, the concentration of each component can be selected from the following ranges or values: about 30-160 mg / ml, about 40-150 mg / ml, about 50-130 mg / ml, about 50-100 mg / ml, about 60-90 mg / ml, about 30-50 mg / ml or about 10-30 mg / ml, for example, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 mg / ml.

[0251] In some embodiments, the concentration of the surfactant in the pharmaceutical formulation is about 0.01-5 mg / ml, for example 0.01-2, about 0.05-1, about 0.05-2, about 0.1-0.5, about 0.1-0.4, about 0.1-0.3, about 0.2-0.4 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 0.05-1 mg / ml, more preferably about 0.1-0.3 mg / ml.

[0252] In some embodiments, the pH of the pharmaceutical formulation is about 5.5-7.5, for example, about 6.0-6.5, such as about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 or about 7.2, more preferably about 6.0-6.5.

[0253] In some embodiments, the pharmaceutical formulation comprises:

[0254] (i) about 20-40 mg / ml of an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof;

[0255] (ii) about 20 mM buffer;

[0256] (iii) about 60-90 mg / ml of a stabilizer; and

[0257] (iv) about 0.1-0.3 mg / ml surfactant.

[0258] In some embodiments, the pharmaceutical formulation comprises:

[0259] (i) about 20-40 mg / ml of an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof;

[0260] (ii) about 20 mM buffer, the buffer being a combination of histidine and histidine hydrochloride or histidine;

[0261] (iii) about 60-90 mg / ml of a stabilizer, said stabilizer being sucrose; trehalose; or a combination of sucrose and mannitol; and

[0262] (iv) about 0.1-0.3 mg / ml polysorbate 80;

[0263] The pH of the pharmaceutical preparation is about 6.0-6.5, preferably about 6.5.

[0264] In some embodiments, the pharmaceutical formulation comprises:

[0265] (i) about 20 mg / ml of an antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof;

[0266] (ii) about 20 mM buffer; the buffer is a combination of histidine and histidine hydrochloride or histidine;

[0267] (iii) about 8% (w / v) stabilizer; the stabilizer is sucrose; trehalose; or a combination of sucrose and mannitol; and

[0268] (iv) about 0.02% (w / v) polysorbate 80;

[0269] The pH of the pharmaceutical formulation is about 6.5.

[0270] In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation.

[0271] In another aspect, the present invention provides a lyophilized pharmaceutical formulation containing an antibody-drug conjugate, wherein the lyophilized pharmaceutical formulation after reconstitution forms or can provide the pharmaceutical formulation as described above.

[0272] In some embodiments, the lyophilized pharmaceutical formulation is prepared by lyophilizing a pharmaceutical formulation as described above.

[0273] In some embodiments, after reconstitution of the lyophilized formulation with a solvent, a liquid formulation with a pH of about 5.5-7.5, preferably 6.0-6.5, for example about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5 is provided.

[0274] In another aspect, the present invention provides a lyophilized pharmaceutical formulation containing an antibody-drug conjugate, comprising (i) about 1%-60% (w / w), preferably about 15%-40% (w / w), such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55% (w / w) of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof;

[0275] (ii) about 0.5%-10% (w / w), preferably about 2%-5% (w / w), e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8% or 9% (w / w), of a buffer;

[0276] (iii) about 20%-90% (w / w), preferably about 50%-85% (w / w), for example 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% (w / w), of a stabilizer; and / or

[0277] (iv) about 0.05%-2% (w / w), preferably about 0.1%-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 a surfactant;

[0278] The antibody-drug conjugate, buffer, stabilizer and / or surfactant are defined herein.

[0279] It should be understood that ranges consisting of two specific values ​​listed herein as endpoints or ranges consisting of a specific value and a range endpoint as a new endpoint are considered in the present invention, as if they were specifically disclosed herein.

[0280] In some embodiments, the lyophilized formulation of the present invention is the formulation disclosed in Example 3. In addition, based on the formulation disclosed in Example 3, embodiments resulting from the concentration values ​​of the components in these formulations fluctuating by 50%, 40%, 30%, 20%, 10% or 5%, and the pH value fluctuating by 5%, 4%, 3%, 2%, 1% or 0.5% are also included in the present application.

[0281] In another aspect, the present invention provides a liquid pharmaceutical preparation containing an antibody-drug conjugate, wherein the liquid pharmaceutical preparation containing an antibody-drug conjugate is obtained by reconstituting the above-mentioned lyophilized pharmaceutical preparation with a solvent, and has a pH of about 5.5-7.5, preferably about 6.0-6.5, preferably about 6.5.

[0282] In some embodiments, the liquid pharmaceutical formulation is preferably for administration by injection or infusion, such as for intravenous injection, subcutaneous injection or intramuscular injection.

[0283] In another aspect, the present invention provides a delivery device comprising the pharmaceutical formulation, lyophilized pharmaceutical formulation or liquid pharmaceutical formulation of the present invention.

[0284] In another aspect, the present invention provides use of the preparation of the present invention (the pharmaceutical preparation, lyophilized pharmaceutical preparation or liquid pharmaceutical preparation as described above) in the preparation of a drug, preferably, the drug is used for preventing or treating tumors;

[0285] More preferably, the tumor is a cancer, such as an epithelial cancer or a cancer of the digestive tract, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.

[0286] It should be understood that, if not specifically stated, when it is a liquid, the preparation of the present invention further comprises a solvent, especially an injectable solvent, including but not limited to, for example, purified water such as ultrapure water, water for injection, sterile water, double distilled water, etc. The solvent that can be used to reconstitute the lyophilized preparation of the present invention includes but is not limited to, for example, purified water such as ultrapure water, water for injection, sterile water, double distilled water, physiological saline, Ringer's solution, glucose injection, etc.

[0287] In some embodiments, the present invention provides a pre-filled syringe comprising the pharmaceutical formulation, lyophilized pharmaceutical formulation or liquid pharmaceutical formulation of the present invention as described above, for intravenous injection, subcutaneous injection or intramuscular injection.

[0288] In another aspect, the present invention provides a method for preparing a pharmaceutical formulation according to the present invention, comprising the following steps:

[0289] i. providing a separated and purified antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, and optionally adding it to an ultrafiltration centrifuge tube for centrifugal concentration;

[0290] ii. a solution of a buffer and a stabilizer is preferably an aqueous solution, wherein the type, concentration and pH of the buffer and the stabilizer are as defined in the pharmaceutical preparation described above,

[0291] iii. ultrafiltration of the antibody-drug conjugate of step i into the solution of step ii,

[0292] iv. adjusting the concentration of the replaced antibody-drug conjugate or its pharmaceutically acceptable salt or solvate to the concentration defined in the above-mentioned pharmaceutical formulation;

[0293] v. adding a surfactant or a solution thereof, preferably an aqueous solution, so that the final concentration of the surfactant is the concentration defined in the above pharmaceutical formulation; and

[0294] vi. Optionally, sterile filtration of the solution of step v.

[0295] In another aspect, the present invention also provides a method for preparing the lyophilized pharmaceutical preparation of the present invention, which, in addition to step iv and optionally step vi described above, further comprises the following steps:

[0296] vii. freeze-drying the solution in step v or vi to obtain a freeze-dried pharmaceutical preparation.

[0297] In another aspect, the present invention also provides a drug combination or a drug combination product,

[0298] The drug combination comprises a drug preparation, a lyophilized drug preparation, or a liquid drug preparation of the present invention, and one or more other therapeutic agents (e.g., therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists), etc.).

[0299] On the other hand, the present invention provides a complete kit, which comprises the pharmaceutical preparation, lyophilized pharmaceutical preparation, or liquid pharmaceutical preparation of the present invention, the pharmaceutical combination of the present invention, preferably the kit is in the form of a pharmaceutical dosage unit. Thus, the dosage unit can be provided according to the dosage regimen or the drug administration interval.

[0300] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0301] - a first container containing the formulation of the invention;

[0302] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0303] In another aspect, the present invention provides a method for preventing or treating a tumor (eg, cancer) in a subject, comprising administering to the subject an effective amount of the formulation, pharmaceutical combination, or kit of the present invention.

[0304] In some embodiments, the tumor (eg, cancer) patient has (eg, elevated levels, such as nucleic acid or protein levels) CLDN18.2. In some embodiments, the patient's tumor cells express CLDN18.2, such as moderate expression of CLDN18.2, preferably high expression of CLDN18.2.

[0305] In some embodiments, the tumor, such as cancer, includes solid tumors and blood tumors and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be in early, middle or late stages or be metastatic cancer.

[0306] In a specific embodiment, the preparation of the present invention can kill tumor cells and / or inhibit the proliferation of tumor cells, such as tumor cells expressing CLDN18.2, such as digestive tract tumor cells, such as gastric cancer cells or pancreatic cancer cells or colon cancer cells or colorectal cancer cells.

[0307] In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface; in some embodiments, the target cell is a CHO cell or 293 cell expressing CLDN18.2, such as a CHO-S cell or HEK293 cell; in some embodiments, the target cell is a cancer cell expressing CLDN18.2, such as a cell that naturally expresses CLDN18.2 or artificially transfects to express CLDN18.2 or artificially transfects to have an increased expression level of CLDN18.2, such as a gastric cancer cell or pancreatic cancer cell line or a colon cancer or colorectal cancer cell line expressing CLDN18.2. In some embodiments, the target cell is a cell line with a medium expression level of hCLDN18.2, such as NUGC-4, SNU620. In some embodiments, the target cell is a cell line with a high expression level of hCLDN18.2, such as a DAN-G cell overexpressing hCLDN18.2.

[0308] In some embodiments, the tumor is an immune evasive tumor.

[0309] In some embodiments, the tumor is a cancer, such as an epithelial tumor, such as a digestive tract tumor, such as an epithelial cancer or a digestive tract cancer, such as gastric cancer or gastroesophageal junction cancer or pancreatic cancer or colorectal cancer or colon cancer.

[0310] The subject can be a mammal, e.g., a primate, preferably, a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or having a risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., cancer) or has a risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and / or radiotherapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0311] In other aspects, the present invention provides the use of the formulation, pharmaceutical combination or kit of the present invention in the production or preparation of a medicament for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0312] In some embodiments, the formulation, pharmaceutical combination or kit of the invention delays the onset of a disorder and / or symptoms associated with a disorder.

[0313] In some embodiments, the formulations of the invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, such as for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0314] In some embodiments, the treatment modality includes surgery; radiation therapy, localized or focused irradiation, and the like.

[0315] In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, an angiogenesis inhibitor, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug, or an immunomodulator (e.g., an immune checkpoint inhibitor or agonist).

[0316] Exemplary additional antibodies include antibodies that specifically bind to immune checkpoints.

[0317] Combination therapies of the invention encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or in separate formulations), and separate administration, in which case administration of the pharmaceutical formulations of the invention can occur prior to, concurrently with, and / or after administration of the other therapeutic and / or agents.

[0318] The composition can also be administered locally via an implanted membrane, sponge, or another suitable material on which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implant device is used, the device can be implanted in any suitable tissue or organ and can deliver the desired molecule via diffusion, a timed-release bolus, or continuous administration.

[0319] In another aspect, the present invention provides a formulation of the invention as described above for use in therapy and / or as a medicament, such as for the uses described herein, such as for the prevention or treatment of a relevant disease or condition mentioned herein.

[0320] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0321] Example 1.1 Synthesis of IEX019 ADC Molecule

[0322] The anti-CLDN18.2 monoclonal antibody HB37A6 was prepared as shown in CN202010570517.X (corresponding to PCT application PCT / CN2021 / 100870). Based on HB37A6, ADCs coupled with different small molecule compounds were further designed and synthesized. The specific process is as follows:

[0323] Example 1.1.1 Preparation of IEX019-02

[0324] 1). Preparation of Compound 6

[0325]

[0326] Under 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 by adding NH4Cl aqueous solution (saturated, 150 mL). After separation, the aqueous layer was extracted with DCM (150 mL). The combined organic layers were dried (Na2SO4) and concentrated. The residue was purified by column chromatography. The product 6 (2.06 g, 5.72 mmol, 57%) was obtained as a light yellow thick 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).

[0327] 2) Preparation of Compound 7

[0328]

[0329] To a stirred solution of 6 (47 mg, 0.13 mmol) in DCM (10 mL) was added CSI (11 μL, 18 mg, 0.13 mmol). 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 another 4.5 hours, the reaction mixture was concentrated and the residue was purified by gradient column chromatography (33→66% EtOAc / heptane (1% AcOH)) to give 7 (88 mg, 0.098 mmol, 75%) as a colorless oil. 1HNMR (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).

[0330] 3) Preparation of Compound 9

[0331]

[0332] 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 DMF (0.9 mL). After 20 hours, the reaction mixture was diluted to 9 mL DCM and purified by gradient column chromatography (0→40% MeOH / DCM) to give 9 (155 mg, 159 μmol, 68%). LCMS (ESI+) C 55 H 54 F6O 10 + (M+H) + Calculated value: 977.39; measured value: 977.80.

[0333] 4) Preparation of Compound 1

[0334] To a solution of compound 9 (155 mg, 159 μmol) in DMF (1.6 mL) was added Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 7 (65 mg, 72 μmol) in DMF (1.4 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 give compound 1 (94 mg, 44 μmol, 28%) as a light 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.

[0335] 5) Enzymatic remodeling of HB37A6 to HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2

[0336] HB37A6 was expressed and purified in HEK293 cells. 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 (equivalent) compared to the antibody) disclosed in PCT / EP2016 / 059194 (in a solution of histidine (20 mM) + NaCl (150 mM) containing 6 mM MnCl2) at 30°C for 16 hours.

[0337] Next, the incubation mixture obtained above was purified using a 50 mL protA column (Hitrap Mabselect Sure, GE, 11-0034-95). The incubation mixture obtained above was loaded onto the column and the column was washed with TBS+0.2% Triton and TBS. The column was then eluted with 0.1 M acetate buffer pH 2.9 and neutralized with 2.5 M Tris-HCl pH 7.2. After dialysis 3 times with PBS, the (modified) glycosylated antibody obtained was concentrated to 32.6 mg / mL using a Vivaspin Turbo 15 ultrafiltration device (Sartorius).

[0338] The obtained glycosylated antibodies were analyzed by mass spectrometry. The main steps were as follows: Before mass spectrometry analysis, the glycosylated antibodies were treated with IdeS so that the Fc / 2 fragment could be analyzed. 20 μg (modified) glycosylated antibody solution was mixed with IdeS (Fabricator TM ) (1.25 U / μL) was incubated in PBS pH 6.6 in a total volume of 10 μL at 37°C for 1 hour. The sample was diluted to 80 μL and then analyzed on a JEOL AccuTOF (ESI-TOF) and the deconvoluted spectra were obtained using Magtran software. Mass spectrometry analysis of the IdeS digested sample showed that the major product corresponded to the obtained HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2, with an observed mass of 24330.4.

[0339] Therefore, the above results demonstrated that HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 in which GlcNAc at Asn297 of both heavy chains was substituted with 6-azido-GalNAc (4-position substitution) was obtained.

[0340] 6) Preparation of HB37A6-SYNtecan E conjugate (IEX019-02)

[0341] The bioconjugate IEX019-02 according to the present invention is prepared by coupling compound 1 (linker-payload) as a linker-conjugate to azide-modified HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 as a biomolecule. Therefore, PBS pH 7.4 (808 μL), 1,2-propylene glycol (11.3 mL) and compound 1 (350 μL, 40 mM DMF solution) were added to HB37A6-(GlcNAc(Fuc)1-6-N3-GalNAc)2 (10.8 mL, 350 mg, 32.6 mg / ml, PBS pH 7.4) solution. The reaction was incubated at room temperature overnight and then filtered and dialyzed to PBS pH 7.4.

[0342] 7.4. Residual free drug was removed by adding charcoal (350 mg) and then rotating overnight at room temperature. The charcoal was removed by centrifugation and filtration, and the ADC was purified on an AKTA Purifier-10 (GE Healthcare) with a Superdex200 26 / 600 SEC (GE Healthcare) column.

[0343] Mass spectrometry analysis of the IdeS digested sample showed two major products, both corresponding to the obtained ADC, HB37A6-SYNtecan E conjugate. The first peak: observed mass 26469 Da (calculated mass 26465 Da), corresponds to the conjugated Fc / 2 fragment (2x closed lactone form of the payload). The second peak: observed mass 26499 Da (calculated mass 26501 Da), corresponds to the bound Fc / 2 fragment (2x open carboxylate form of the payload).

[0344] 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 >99% and the concentration was 6.12 mg / ml.

[0345]

[0346] Wherein q represents the average DAR, for example, 3-5, 3.2-4.8, or 3.5-4.5, such as 3.52 determined in Table 1.

[0347] Among them, Ab is HB37A6.

[0348] Example 1.1.2 Preparation of IEX019-03

[0349]

[0350] wherein q represents the average DAR, for example, 5-11, 6-10, 7-9 or 7.5-8.5, such as 7.9 as determined in Table 1.

[0351] Among them, Ab is HB37A6.

[0352] (a) A reducing agent solution (TCEP (Sigma, C4706), dissolved in water) was added to an HB37A6 solution (antibody HB37A6 was dissolved in PBS buffer (Thermo, 10010023)), and the reaction mixture was placed in an oscillator for 2-4 h.

[0353] (i) The optimal concentration of HB37A6 is 5-10 mg / mL.

[0354] (ii) The optimal molar ratio of TCEP / mAb is 4.5-6.5.

[0355] (iii) The optimal temperature for the reaction is 20-37°C.

[0356] (iv) The optimum pH value for the reaction is generally between 6.5 and 8.0.

[0357] (b) dissolving an excess of linker-toxin MC-GGFG-DXD (purchased from Levena biopharma, SET0218, the structure of which can be shown below) in DMSO and reacting with the thiol group of the antibody reduced in step (a). The reaction mixture is placed in an oscillator for 1-2 hours.

[0358] (i) The optimal molar ratio of DXD / mAb is 10.0-12.0,

[0359] (ii) The optimal reaction temperature is 20-37°C,

[0360] The crude ADC product was obtained.

[0361] (c) The obtained ADC crude product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IEX019-03.

[0362] (d) The ADC products were tested by HIC, LC-MS and SEC HPLC to determine the average DAR and SEC purity.

[0363]

[0364] Example 1.1.3 Preparation of IEX019-04

[0365]

[0366] wherein q represents the average DAR, for example 3-5, 3.2-4.8 or 3.0-4.0, such as 3.5 as determined in Table 1.

[0367] Among them, Ab is HB37A6.

[0368] The preparation of this molecule was carried out as follows.

[0369] (a) Add a reducing agent solution (TCEP (Sigma, C4706), dissolved in water) to an antibody HB37A6 solution (antibody HB37A6 dissolved in PB buffer). After the addition is complete, place the reaction mixture in an oscillator for 2-4 hours.

[0370] (i) The optimal concentration of antibody HB37A6 is 5-10 mg / mL

[0371] (ii) The optimal molar ratio of TCEP / mAb was 1.9-2.7.

[0372] (iii) The optimal temperature for the reduction reaction is 20-37°C.

[0373] (iv) The optimum pH value for the reaction is generally between 6.5 and 8.0.

[0374] (b) An excess of the linker-payload MC-VC-PAB-MMAE (purchased from Levena Biopharma, SET0201) with a reactive group (maleimide-linked drug) was dissolved in an organic solvent DMSO and reacted with the reduced thiol group produced in step (a). The reaction mixture was placed in an oscillator for 1-2 h.

[0375] (i) The optimal molar ratio of MC-VC-PAB-MMAE / mAb is 8.0-10.0.

[0376] (ii) The optimal temperature for the binding reaction is 20-37°C.

[0377] (c) After the binding reaction is completed, acetylcysteine ​​solution is added to terminate the reaction of step (b). After mixing, incubate at 20-25°C for 5-15 minutes.

[0378] (d) The obtained ADC crude product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IEX019-04.

[0379] (e) The ADC product was tested by HIC and SEC-HPLC to determine the average DAR and SEC purity.

[0380]

[0381] Example 1.1.4 Preparation of IEX019-05

[0382]

[0383] Wherein q represents the average DAR, for example, 3-5, 3.2-4.8, or 3.0-4.5, such as 3.3 determined in Table 1,

[0384] Among them, Ab is HB37A6,

[0385] This molecule was prepared as follows:

[0386] (a) Add the linker-payload SMCC-DM1 solution (purchased from Levena Biopharma, SET0101, dissolved in an organic solvent such as DMSO) to the antibody HB37A6 solution (the antibody HB37A6 is dissolved in PB buffer), and after the addition, place the reaction mixture in an oscillator for 2-5 hours, wherein:

[0387] (i) The optimal concentration of antibody HB37A6 is 5-10 mg / mL;

[0388] (ii) the optimal molar ratio of linker-payload / mAb was 5.5–6.5;

[0389] (iii) The optimal pH value of the reaction is generally between 6.5 and 8.0;

[0390] (iv) The optimal temperature for the reaction is 20-37°C;

[0391] Obtain ADC crude product,

[0392] (b) The obtained ADC crude product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product IEX019-05.

[0393] (c) The DAR average value and SEC purity of the ADC product were determined by UV spectrophotometry and SEC high performance liquid chromatography.

[0394]

[0395] Example 1.1.5 Preparation of IEX019-06

[0396] The preparation process is similar to that of IEX019-02, except that the HB37A6 monoclonal antibody is replaced with the control antibody IgG.

[0397] Example 1.1.6 Preparation of IEX019-07

[0398] The preparation process is similar to that of IEX019-04, except that the HB37A6 monoclonal antibody is replaced with the control antibody IgG.

[0399] The information of all mAbs and ADCs is summarized in Table 1 .

[0400] Table 1. IEX019 molecular information

[0401]

[0402]

[0403] *The heavy chain of IgG is SEQ ID NO: 13, and the light chain is SEQ ID NO: 14.

[0404] Example 1.2 Cell binding experiment of IEX019 molecule

[0405] In order to detect whether small molecule conjugation will change the binding properties of IEX019-01 monoclonal antibody with target cells, we used DAN-G cell line (hCLDN18.2 negative) and DAN-G-hCLDN18.2 cell line (overexpressing hCLDN18.2) prepared in Example 1 of CN202010570517.X to detect the affinity of IEX019-01 and IEX019-02 to the target by flow cytometry (FACS). The specific experimental method is as follows.

[0406] The cells to be tested (CHO-hCLDN18.2) were counted and diluted to 1×10 6Cells / ml, add 100μl / well to a U-bottom 96-well plate. 500g 5min, centrifuge, and remove the cell culture medium. Add molecules IEX019-01 and IEX019-02 and control molecule IEX019-06 to the U-shaped plate and resuspend the cells, add 100μl to each well, the starting concentration of the molecule is 900nM, and then dilute 3 times in sequence, for a total of 10 concentration points. Stand on ice for 30min. 500g 5min remove the supernatant, wash the cells once with PBS. Add 100μl of goat anti-human Fc PE-labeled secondary antibody (SouthernBiotech, J2815-5H87B) to each well. Incubate on ice in the dark for 30min. 500g 5min remove the supernatant, wash the cells once with PBS. Resuspend the cells with 50μl 1×PBS and detect on FACS machine. Use GraphPad Prism software to analyze the experimental data.

[0407] IEX019-01 and IEX019-02 did not bind to non-target cell DAN-G, but had a strong affinity to DANG-hCLDN18.2, indicating that the binding of the antibody depends on the expression specificity of the target, and the coupling of Exatecan does not affect the binding of the antibody. At the same time, the control molecule IEX019-06 (the monoclonal antibody is a negative control for IgG, and IgG is coupled to Exatecan toxin using the same technology) does not bind to the target cell ( Figure 1 ).

[0408] Example 1.3 IEX019 molecule endocytosis experiment

[0409] Strong endocytosis is one of the important characteristics of ADC drugs. When ADC binds to the antigen on the cell membrane surface, the ADC-antigen complex enters the cell through endocytosis and kills the target cell. Therefore, the endocytosis efficiency of ADC is one of the important indicators that determine the tumor suppression effect.

[0410] In order to detect the internalization efficiency of antibodies after coupling with small molecule compounds, we used flow cytometry to detect the internalization of different IEX019 molecules in DANG-hCLDNA18.2 cells. After DANG-hCLDNA18.2 cells were digested, the cell density was adjusted to 1*10 5100 μl of the molecule to be detected was resuspended in the cells (molecular concentration was 50 nM), and 5 replicates were set for each sample (i.e., endocytosis time: 0h, 1h, 2h, 3h, 4h). Place on ice and incubate for 1h. After 1h, centrifuge at 500g for 3min and discard the supernatant. Add 200μl FACS buffer (1% FBS, 1XPBS) to each well and rinse twice. Take one of the samples, transfer it to a new 96-well plate, and incubate at 37℃ for 4h. The remaining samples continue to incubate on ice. Repeat the previous operation and take samples to incubate at 37℃ for 3h, 2h, 1h, and 0h. After the specific incubation time is completed, centrifuge at 500g for 3min and discard the supernatant. Add 100μl of 1:400 diluted anti-hFC-PE antibody (SouthernBiotech) to each well and incubate on ice for 30 minutes in the dark. After the secondary antibody incubation, centrifuge at 500g for 3 minutes and discard the supernatant. Add 200μl FACS buffer to each well for two rinses, and finally resuspend the cells with 100μl PBS for detection.

[0411] The experimental results are as follows Figure 2 As shown, taking 0h of incubation at 37°C as the zero point of endocytosis, after 2h of incubation, all molecules reached the maximum limit of endocytosis, about 60%, indicating that after the ADC molecules designed and synthesized based on IEX019-01 bind to tumor cells, they maintain a strong ability to be internalized, consistent with the monoclonal antibody.

[0412] Example 1.4 In vitro cell killing effect of IEX019 molecule

[0413] The CellTiter-Glo (Promega, G9242) detection kit was used to detect the effect of ADCs on cell viability in various cell lines expressing different levels of hCLDNA8.2 (Table 2).

[0414] Table 2. Expression of hCLDN18.2 in the cell lines used

[0415]

[0416] After the cells were digested with EDTA / Trypsin, the density was adjusted and evenly spread into a 96-well plate (Table 3), and the diluted sample IEX019 molecules of specific concentrations (IEX019-02, IEX019-03, IEX019-04, the starting concentration of dilution was 100nM, and the dilution factor was 3) were added. The wells without IEX019 molecules were used as controls. Place in a 37°C incubator and incubate for 5 days. After 5 days, 100μl CellTiter-Glo detection reagent was added to each well, incubated at room temperature for 30min, and detected with an enzyme marker. Calculate the relative cell viability (relative cell viability = sample / control * 100%) and fit the curve using Graph Pad Prism 8.0.

[0417] like Figure 3 As shown in Figure 2, the killing of cell lines by ADC molecules depends on the expression level of surface hCLDN18.2. In hCLDNA8.2-negative DANG, IEX019 molecules had no significant effect on cell viability ( Figure 3 A); In cell lines with medium expression levels (NUGC-4, SNU620), IEX019 molecules have a certain degree of cell killing ( Figure 3 B); IEX019 molecules have significant killing effects on the high-expression cell line DAN-G-hCLDN18.2 ( Figure 3 C). This indicates that the IEX019 molecule has good selectivity and effectiveness.

[0418] Table 3. Cell seeding density

[0419]

[0420] Example 1.5: Bystander killing effect

[0421] During the synthesis of ADC drugs, small molecule compounds can be connected to antibodies through cleavable linkers. After being internalized into the cell membrane, the linker is cleaved and the small molecule is released to kill 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 called the bystander effect. Since cells inside the tumor can have great differences in target expression levels (tumor heterogeneity), the bystander effect is very important for effectively killing tumor cells and inhibiting tumor growth.

[0422] The present invention utilizes non-target cell DAN-G and target cell DANG-hCLDN18.2 to detect the bystander effect of the IEX019 molecule.

[0423] After the cells were digested with EDTA / Trypsin, the density was adjusted, a six-well cell culture plate was prepared, and 7.5×10 DANG cells and 7.5×10 DANG-hCLDN18.2 were added. 4 , and the two types of cells were co-cultured. 200 μl of the sample to be tested (IgG (SEQ ID NO: 13, SEQ ID NO: 14), IEX019-02, IEX019-05, IEX019-06, Exatecan (Macklin, E881532)) was added to each well, with a final concentration of 50 nM, and 3 replicates were set for each sample. The cell culture plate was placed in a 37°C incubator and cultured for 5 days. After 5 days, the culture supernatant was discarded, and Trypsin-EDTA was added to digest the cells after rinsing with PBS. All digested cells were collected and transferred to a 96-well plate. According to the antibody incubation process of flow cytometry detection technology, incubated with the primary antibody (IEX019-01, 100 nM) and the secondary antibody (anti-hFc-PE, Southern Biotech) at 4°C for 1h and 0.5h respectively. After rinsing with PBS, the live / dead Violet dye (Thermo, L34964) was diluted 1:1000, 100 μl was added to each well, and incubated at 4°C for 20 min. After rinsing with PBS, the cells were resuspended in 100 μl PBS and tested on the machine. Live / dead dye was used to separate the populations in each sample, where the IEX019-01 negative (i.e., hCLDN18.2 negative) population was DAN-G cells, and the IEX019-01 positive (i.e., hCLDN18.2 positive) cells were DAN-G-hCLDN18.2. The number of the two types of cells in each sample was counted separately, and the relative cell viability of each cell was calculated according to the following equation, and the curve was fitted using Graph Pad Prism 8.0.

[0424] Relative viability of DANG cells = number of DNAG cells in sample group / number of DNAG cells in IgG group*100%

[0425] Relative viability of DANG-hCLDN18.2 cells = number of DNAG-18.2 cells in sample group / number of DNAG-18.2 cells in IgG group * 100%

[0426] The cell viability of the IgG group was set as 100%.

[0427] like Figure 4As shown, the negative control sample IEX019-06 had no killing effect on both cells; the IEX019-05 molecule connected the DM1 toxin to the IEX019-01 monoclonal antibody through an uncuttable linker and had no ability to kill surrounding non-target cells, so it could only kill DANG-hCLDNA8.2 cells, but had no effect on DANG cells. Only IEX019-02 had a significant bystander effect, which could kill both target cells and non-target cells.

[0428] Example 1.6: Antitumor efficacy of IEX019 in the DAN-G-hCLDN18.2 mouse transplant tumor model

[0429] In order to demonstrate the in vivo efficacy of the IEX019 molecule, we used DANG-hCLDN18.2 cells to inoculate CB-17-SCID mice to determine the anti-tumor efficacy of the antibody molecule of the present invention. The experiment used SPF-grade female CB-17-SCID mice (14-17 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and the certificate number was NO.110011201108225246.

[0431] DANG-hCLDN18.2 cells were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation and dispersed with PBS (1×) to prepare a cell concentration of 3×10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB-17 SCID mice to establish the DANG-hCLDN18.2 tumor-bearing mouse model.

[0432] On the fifth day after tumor cell inoculation, the tumor volumes of all mice ranged from 50.16 to 136.68 mm 3 The mice were divided into groups (6 mice per group) and the dosage and method of administration were as shown in Table 4. The drugs were administered on the 5th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. Figure 5 As shown in A and 5B, the monitoring ended after 92 days.

[0433] The relative tumor inhibition rate (TGI%) was calculated on the 50th day after inoculation, and the calculation formula was as follows:

[0434] TGI%=100%*(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume of control group before drug administration).

[0435] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula:

[0436] V=L*W 2 / 2. Body weight was measured using an electronic balance.

[0437] Table 4. Experimental design table (Note: single administration, 1 time in total)

[0438] Group Dosage Dosing frequency Dosage IEX019-01 (control group) 10mg / kg Single dose Intraperitoneal injection IEX019-02 (treatment group) 10mg / kg Single dose Intraperitoneal injection IEX019-03 (treatment group) 10mg / kg Single dose Intraperitoneal injection IEX019-04 (treatment group) 10mg / kg Single dose Intraperitoneal injection

[0439] The tumor inhibition rate results are shown in Table 5 and Figure 5 As shown in A: On the 50th day after inoculation, the tumor inhibition rate of IEX019-02 reached 103.60% after a single dose compared with IEX019-01 monoclonal antibody, which was significantly better than IEX019-03 and IEX019-04, with tumor inhibition rates of 93.70% and 35.20%, respectively. 82 days after inoculation, 100% of the mice in the IEX019-02 group had complete tumor regression. At the same time, we tested the weight of the mice, and the results are as follows Figure 5 There was no significant difference in the body weight of mice shown in B.

[0440] Table 5. Tumor inhibition rate on day 50

[0441]

[0442] Example 1.7: Antitumor efficacy of IEX019 in the NUGC-4 mouse transplant tumor model

[0443] In order to demonstrate the in vivo efficacy of the IEX019 molecule, we used NUGC-4 cells to inoculate CB-17-SCID mice to determine the anti-tumor efficacy of the antibody molecule of the present invention.

[0444] The experiment used SPF-grade female CB-17-SCID mice (14-17 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), with the certificate number NO.110011201109348141.

[0445] NUGC-4 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS (1×) to prepare a cell concentration of 3×10 7 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB-17 SCID mice to establish a NUGC-4 tumor-bearing mouse model.

[0446] On the fifth day after tumor cell inoculation, the tumor volume of all mice was 72.25-140.50 mm 3 The mice were divided into groups (6 mice per group) and the dosage and method of administration were as shown in Table 6. The mice were given the fifth dose after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. Figure 6 As shown in A and 6B, the monitoring ended after 40 days.

[0447] The relative tumor inhibition rate (TGI%) was calculated on the 33rd day after inoculation, and the calculation formula was as follows:

[0448] TGI%=100%*(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume of control group before drug administration).

[0449] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula:

[0450] V=L*W 2 / 2. Body weight was measured using an electronic balance.

[0451] Table 6. Experimental design table (Note: single administration, 1 time in total)

[0452]

[0453]

[0454] The tumor inhibition rate results are shown in Table 7 and Figure 6 As shown in A: On the 33rd day after inoculation, the tumor inhibition rates of IEX019-02 and IEX019-03 were 80.04% and 54.31% respectively compared with the negative control IEX019-06. At the same time, we tested the weight of mice. The results are shown in Figure 6 There was no significant difference in the body weight of mice shown in B.

[0455] Table 7. Tumor inhibition rate on day 33

[0456]

[0457] Example 1.8: Antitumor efficacy of IEX019 in the SNU620 mouse transplant tumor model

[0458] In order to demonstrate the in vivo efficacy of the IEX019 molecule, we used SNU620 cells to inoculate CB-17-SCID mice to determine the anti-tumor efficacy of the molecule of the present invention (IEX019-02). The experiment used SPF-grade female CB-17-SCID mice (18-20 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and the certificate number was NO.110011211102179364.

[0460] SNU620 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS (1×) to prepare a cell concentration of 3×10 7On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal area of ​​CB-17 SCID mice to establish the SNU620 tumor-bearing mouse model.

[0461] On the 7th day after tumor cell inoculation, the tumor volume of all mice was 58.1-117.3 mm 3 The mice were divided into groups (6 mice per group) and the dosage and method of administration were as shown in Table 8. The mice were administered on the 7th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. Figure 7 As shown in A and 7B, the monitoring ended after 39 days.

[0462] The relative tumor inhibition rate (TGI%) was calculated on the 39th day after inoculation, and the calculation formula was as follows:

[0463] TGI%=100%*(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume of control group before drug administration).

[0464] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula:

[0465] V=L*W 2 / 2. Body weight was measured using an electronic balance.

[0466] Table 8. Experimental design table (Note: single administration, 1 time in total)

[0467]

[0468] The tumor inhibition rate results are shown in Table 9 and Figure 7 As shown in A: On the 39th day after inoculation, compared with hIgG, the tumor inhibition rate after a single dose of IEX019-02 10 mg / kg was 143.77%, and 100% of the mice had complete tumor regression. There was no significant difference in the weight of mice in each treatment group compared with the hIgG control group ( Figure 7 B).

[0469] Table 9. Tumor inhibition rate on day 39

[0470]

[0471]

[0472] Example 2 Prescription Study of IEX019-02 Preparation

[0473] 1. Overview

[0474] This experiment aims to develop a long-term stable formulation of IEX019-02. The development of this formulation includes two steps: 1) pH buffer screening (4 weeks at 40°C pressure conditions). 2) Excipient screening, including liquid samples placed at 25°C and 40°C pressure conditions for 4 weeks, 5 freeze-thaw cycles, and oscillation for 3 days (150rpm, 25°C). Lyophilized samples were placed at 40°C for 2 weeks, 4 weeks, and 12 weeks. Analytical methods include DSC, appearance, pH, ADC molecular concentration, insoluble particles (HIAC), SE-HPLC, iCIEF, Caliper-R / NR, DAR value, free drug, reconstitution time, and binding activity.

[0475] 2. Overview of formulation analysis methods

[0476] 2.1 Appearance

[0477] Check the appearance of the sample, including clarity, color, and visible foreign matter, against a black and white background using a YB-2 clarity tester.

[0478] 2.2 pH value

[0479] The pH of the samples was measured using a pH meter with a glass electrode. The pH meter was calibrated with three different standard buffers (pH 4.01, 7.00, and 9.21) before the first use of each day. The calibration slope was between 95.0% and 105.0%, and the zero drift was between -60.0mv and +60.0mv.

[0480] 2.3 ADC Molecular Concentration

[0481] Nanodrop or solo-VPE is used to detect the absorbance of ADC at 280nm and 365nm, and the ADC molecular concentration is calculated according to the following formula. CF is the correction factor, AWV is the absorbance at 365nm, and EC is the extinction coefficient (1.52Au*mL*mg-1*cm-1)

[0482]

[0483] 2.4 Insoluble particles (HIAC)

[0484] The size and count of insoluble particles were measured using a HIAC particle analyzer (HIAC 9703+) under a laminar flow hood. The sample was tested using 0.45 mL of sample at a time and performed 4 times in succession. The first run test result was discarded and the average of the number of insoluble particles per milliliter in the last 3 runs (insoluble particle size ≥ 2 / 10 / 25 μm) was reported.

[0485] 2.5 Size Exclusion Chromatography Purity SE-HPLC

[0486] SE-HPLC is a purity analysis method that separates proteins according to their size. The SE-HPLC analysis procedure is as follows: Detection was performed on an Agilent 1260 HPLC system equipped with a TSKgel G3000SWXL column (7.8×300 mm, 5 μm particle size) and a UV detector (detection wavelength: 280 nm). The sampler temperature was set to 5±3°C, and the column oven temperature was set to 25±3°C. The mobile phase was 0.078 M potassium dihydrogen phosphate, 0.122 M potassium dihydrogen phosphate trihydrate, 0.15 M potassium chloride, and 15% (v / v) IPA, with a pH of 7.0±0.1, and the flow rate was set to 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 run time was 20 minutes. Finally, all raw data were processed using CDS software.

[0487] 2.6 Drug-drug conjugation ratio DAR

[0488] The DAR analysis procedure was as follows: the detection was performed on a Waters ultra-high performance liquid chromatography UPLC system equipped with a Waters / ACQUITY UPLC Protein BEH C4 Column, Column (2.1×150mm, 1.7μm particle size) and UV detector (detection wavelength: 280nm). The sampler temperature was set to 5±3℃, and the autosampler temperature was set to 70±1℃. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution containing 5% IPA; mobile phase B was: 0.1% trifluoroacetic acid acetonitrile solution containing 5% IPA. The flow rate was 0.25mL / min. 10μg of each sample was injected into the HPLC vial. The detection wavelength was set to 280nm and the running time was 45 minutes. Finally, all raw data were processed using Empower software.

[0489] 2.7 Free drug

[0490] The Free Drug analysis procedure is as follows: Detection was performed on an Agilent Technologies 1260 system equipped with an Agilent Eclipse Plus C18 column (4.6×100 mm, 3.5 μm particle size) and a UV detector (detection wavelength: 365 nm). The injector temperature was set to 5±3°C and the column temperature was set to 50±1°C. Mobile phase A was 0.1% trifluoroacetic acid in water; 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.

[0491] 2.8 Charge isomerization iCIEF

[0492] The sample was diluted to 2.0 mg / ml with ultrapure water, and then 20 μL of the diluted sample was mixed with 80 μL of the premix to prepare a loading mixture with a final concentration of 0.4 mg / mL. The premix consisted of 4.0 μL of ampholytes 3-10, 35 μL of 1% methylcellulose, 0.5 μL of isoelectric point marker pI 7.05, 0.5 μL of isoelectric point marker 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. The detection was completed within 48 hours after the sample and premix were mixed. The loading solution was analyzed using an iCE3 capillary isoelectric focusing analysis system equipped with an FC-coated full-column imaging capillary. After separation, the raw data was processed using Water / Empower FR5.0.

[0493] 2.9 Reducing / non-reducing capillary gel electrophoresis purity Caliper-NR / R

[0494] Non-reduced Caliper-SDS was analyzed on a SCIEX / PA800 Plus instrument equipped with a PDA detector (diode array detector). The sample was diluted to 2.0 mg / mL. Take 50 μL of sample, place it in a 1.5 mL centrifuge tube, take 5 μL of NEM (N-ethylmaleimide) and add it to the centrifuge tube, and then add 45 μL of SDS sample buffer (PB-CA (citrate phosphate buffer) containing 0.8% SDS). After mixing and centrifuging, place the sample at 70°C for 10 minutes. The capillary right end injection method was used (the effective separation length of the capillary is 20.2 cm). The detection wavelength is 220 nm. After continuous injection for 20 seconds using a forward voltage of -5 kV, continue to separate for 35 minutes using a forward voltage of +15 kV.

[0495] Reduced Caliper-SDS was analyzed on a SCIEX / PA800 Plus instrument equipped with a PDA detector. The sample was diluted to 2.0 mg / mL. Take 50 μL of sample, place it in a 1.5 mL centrifuge tube, and add 45 μL of pH 6.5 sample buffer (PB-CA (citrate phosphate buffer) containing 0.8% SDS) and 5 μL of BME (β-mercaptoethanol). After mixing and centrifuging, the sample was placed at 70°C for 10 minutes, and then the sample was centrifuged at 13000 rpm in a centrifuge for about 1 minute. The capillary left end injection method was used (the effective separation length of the capillary is 20.2 cm). The detection wavelength is 220 nm. Use a reverse voltage of -5 kV to continuously inject for 20 seconds, and continue to use a reverse voltage of -15 kV for separation for 35 minutes.

[0496] 2.10 Binding activity

[0497] The reference, control and test products with gradient dilutions are added to a 96-well high binding plate, and the products bind to the antigens coated on the plate. After washing, a peroxidase-labeled goat anti-human IgG Fcγ detection antibody (Jackson, 109-035-098#) is added to the plate. The detection antibody can bind to the captured ADC product. After the final wash, TMB (3,3',5,5'-tetramethylbenzidine) substrate solution is added to the plate. TMB can specifically react with peroxide in the presence of peroxidase to generate a single color signal, and the signal intensity is proportional to the amount of ADC product bound to the plate. After terminating the color reaction, read the absorbance value 450nm-620nm on the microplate reader.

[0498] The reference, control and test sample curves were drawn using a four-parameter fitting regression model using SoftMax Pro software to obtain the EC50 of the reference, control and test samples in the same plate. The relative binding activity of the control and test samples was calculated using the following formula:

[0499] The relative binding activity of the control or test article = EC50 of the reference article / EC50 of the control or test article × 100%.

[0500] 2.11 Reconstitution time

[0501] Take a 10 ml syringe, draw more than 5.2 ml of water for injection, and remove the air so that the final volume of water for injection in the syringe is 5.2 ml. Insert the syringe from the center of the rubber stopper, add water for injection along the bottle wall, and press the timer. Grasp the aluminum cap of the bottle mouth with your hand, and rotate your wrist slightly clockwise. If no particles are observed within 10 minutes, press the timer again, and this time is the re-dissolution time.

[0502] 2.12DSC

[0503] DSC is a thermal analysis instrument that measures the difference in heat between a sample and a reference when heated. This experiment was measured using a MicroCal PEAQ DSC (Malvern, SYS12907). Prior to analysis, the protein sample was first diluted to 1 mg / mL using its reference buffer. 400 μL of each reference buffer was added to the odd-numbered wells of a 96-well plate, and 400 μL of sample was added to the even-numbered wells of the same plate. The experimental parameters were set so that the scanning temperature increased from 10°C to 95°C at a rate of 90°C / h. Thermospectral analysis was then performed using the MicroCal PEAQ DSC automatic data analysis software.

[0504] 2.13 Glass transition temperature detection (Tg')

[0505] The glass transition temperature (Tg') of the protein formulation is determined by TA 2000DSC. 20μL of sample is placed in an aluminum sample pan, and then the sample pan with sample and an empty sample pan (reference) are placed in the DSC instrument. Set the corresponding parameters in the control panel (parameters are shown in the table below), including heating / cooling rate, heating / cooling target temperature, and platform insulation time after reaching the target temperature. After the settings are completed, start data collection. The mDSC spectrum is recorded during this process, and the glass transition temperature (Tg') is obtained by Universal Analysis.

[0506] Heating / cooling rate temperature Maintenance time (min) Pre-freeze 1 10℃ / min -50℃ 3min annealing 10℃ / min -7℃ 5min Pre-freeze 2 10℃ / min -50℃ 3min Warming up 5℃ / min 20℃ NA

[0507] 2.14 Collapse temperature (Tc)

[0508] The collapse temperature (Tc) was measured using a Lyostat5 LED freeze-drying microscope. When testing the sample, place the sample holder on the temperature control block and close the side door of the sample cell to ensure that the sample holder is in the center of the temperature control block. Use a vacuum pen to place a 16mm diameter quartz glass piece on the sample holder so that it is in contact with the temperature control block. Then use tweezers to pick up a 70μm thick metal gasket and place it on the quartz slide, keeping the metal gasket coaxial with the center of the quartz plate. Then use a pipette to draw a certain volume (usually 2μL) of the sample to be tested and drop it in the center of the quartz plate. Use a vacuum pen to carefully place a 13mm diameter cover glass on the metal gasket, ensuring that the edge of the sample moves above the light-transmitting hole of the temperature control block for observation of the sample. Screw the lid back onto the platform and fix the nitrogen outlet tube to the lid to avoid frost. Then set the corresponding parameters in the control panel (see the table below for parameters), including heating / cooling rate, heating / cooling target temperature, platform insulation time after reaching the target temperature, and image acquisition frequency. After the settings are completed, start collecting data. During the freeze-drying process, the sublimation front moves from the outer edge of the sample to the middle. The front of the sublimation front is in a frozen state, and the back is in a dry state. When the sample temperature is higher than the collapse temperature (Tc), it will cause collapse. Starting with the photos with lower temperatures, carefully review all the photos in the experiment. When tiny voids appear in the collected photos, it means that the microstructure has collapsed, and the corresponding temperature is judged as the collapse temperature.

[0509]

[0510]

[0511] 3. pH buffer screening

[0512] 3.1 Experimental Purpose

[0513] This experiment aims to screen the optimal pH buffer system when the concentration of IEX019-02 is 40 mg / mL.

[0514] 3.2 Experimental plan

[0515] As shown in Table 10, 8 sets of experiments with 3 buffer systems were studied at 40 mg / mL IEX019-02 concentration.

[0516] The stability of 8 groups of experiments with 3 different pH buffer systems was evaluated for 4 weeks under high temperature conditions (40°C). The tests conducted in this study included DSC, appearance, pH, ADC molecular concentration, SE-HPLC, Caliper-NR / R, Free drug, DAR value and iCIEF.

[0517] Table 10. ADC pH buffer screening protocol

[0518]

[0519] 3.3 Sample preparation

[0520] Take the IEX019-02 stock solution (10 mg / mL ADC, 20 mM histidine solution, 8% (w / v) sucrose, pH 6.0) and concentrate it with an ultrafiltration tube, then change the solution to 20 mM citric acid buffer (pH 5.0, 6.0), 20 mM succinic acid buffer (pH 5.0, 5.5, 6.0) and 20 mM histidine buffer (pH 5.5, 6.0, 6.5) respectively, adjust the IEX019-02 concentration to about 40 mg / mL, filter the solution with a 0.22 μm PES syringe filter in a biological safety cabinet, and obtain the samples of Table 10F1-F8, which are filled into 2 mL vials with a filling volume of 1 mL / bottle. Immediately after filling, the vials are stoppered and capped. Place the samples under different storage conditions according to Table 10, and place at least 1 bottle of each prescription at each sampling point.

[0521] 3.4 Results and Discussion

[0522] The results of the pH buffer screening for the IEX019-02 formulation are summarized in Tables 11-20.

[0523] 3.4.1DSC

[0524] As shown in Table 11 and Figure 8 As shown, the Tm onset values ​​of the samples in 8 pH buffer systems (F1-F8) were all over 58°C, indicating that the molecule had good conformational stability.

[0525] Table 11. Summary of DSC results for ADC pH buffer screening

[0526]

[0527] 3.4.2 Appearance

[0528] At T0, the samples were pale yellow, slightly opalescent liquids with a small amount of protein particles in all eight pH buffer systems (F1-F8). After being placed at 40°C for 1 or 2 weeks, a large number of 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), and a small amount 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 placed at 40°C for 4 weeks, all samples showed a large amount of particles, but F1-F4 had relatively more particles.

[0529] 3.4.3 pH value

[0530] As shown in Table 12, compared with T0, after being placed at 40°C for 4 weeks, no significant changes in pH values ​​were observed in the other pH buffer systems (F1-F6, F8) except for the histidine 6.0 group (F7).

[0531] Table 12. Summary of pH results for IEX019-02 pH buffer screening

[0532]

[0533] 3.4.4 IEX019-02 Molecular Concentration

[0534] As shown in Table 13, compared with T0, no significant changes in the IEX019-02 concentrations were observed in the samples in the eight pH buffer systems (F1-F8) after being placed at 40°C for 4 weeks.

[0535] Table 13. Summary of IEX019-02 concentration results for pH buffer screening

[0536]

[0537] Note: In this experiment, the concentration of IEX019-02 was detected by Nanodrop.

[0538] 3.4.5 Size Exclusion Chromatography Purity SE-HPLC

[0539] As shown in Table 14, compared with T0, after being placed at 40°C for 1 week, 2 weeks and 4 weeks, the SE-HPLC monomer of 8 pH buffer systems (F1-F8) decreased significantly. At 1 week, the SE-HPLC monomer decreased by 1.7% to 23.1%, and the SE-HPLC monomer of F6-F8 (histidine buffer system, pH 5.5, pH 6.3 and pH 6.5) decreased by 1.7% to 2.6%. At 2 weeks, the SE-HPLC monomer decreased by 8.2% to 30.2%, and the SE-HPLC monomer of F6-F8 (histidine buffer system, pH 5.5, pH 6.3 and pH 6.5) decreased by 8.2% to 10.5%. At 4 weeks, the SE-HPLC monomer decreased by 9.4% to 56.2%, and the SE-HPLC monomer of F6-F8 (histidine buffer system, pH 5.5, pH 6.3 and pH 6.5) decreased by 9.4% to 10.8%, which was less than other pH buffer systems. Therefore, histidine buffer is superior.

[0540] Table 14. Summary of SE-HPLC results for pH buffer screening

[0541]

[0542] 3.4.6 Charge isomerization iCIEF

[0543] As shown in Table 15, compared with T0, after being placed at 40°C for 4 weeks, the main peak of iCIEF in 8 pH buffer systems (F1-F8) decreased by 11.6%-21.8%. The main peak of iCIEF in F7 (histidine buffer system, pH 6.3), F8 (histidine buffer system, pH 6.5), and F5 (succinic acid buffer system, pH 6.0) decreased by 11.6%-17.1%, which was less than that in other pH buffer systems (iCIEF main peak decreased by 18.6%-21.8%). Therefore, F7, F8, and F5 are better.

[0544] Table 15. Summary of iCIEF results for pH buffer screening

[0545]

[0546]

[0547] 3.4.7 Reduction capillary electrophoresis purity Caliper-R

[0548] As shown in Table 16, compared with T0, after being placed at 40°C for 4 weeks, the purity of Caliper-R in 8 pH buffer systems (F1-F8) decreased by 1.0%-4.6%. The purity of Caliper-R in F5 (succinic acid buffer system, pH 6.0) and F6-F8 (histidine buffer systems, pH 5.5, pH 6.3 and pH 6.5) decreased by 1.0%, which was lower than that in other pH buffer systems (Caliper-R purity decreased by 1.4%-4.6%). Therefore, F5, F6, F7, and F8 performed better.

[0549] Table 16. Summary of Caliper-R Results for pH Buffer Screening

[0550]

[0551] 3.4.8 Non-reducing capillary electrophoresis purity Caliper-NR

[0552] As shown in Table 17, compared with T0, after being placed at 40°C for 4 weeks, the purity of Caliper-NR in 8 pH buffer systems (F1-F8) decreased by 3.6%-8.5%. The purity of Caliper-NR in F2 (citrate buffer system, pH 6.0) decreased by 3.6%, which was less than that of other pH buffer systems (Caliper-NR purity decreased by 4.0%-8.5%).

[0553] Table 17. Summary of Caliper-NR results in pH buffer screening

[0554]

[0555] 3.4.9 Free drug

[0556] As shown in Table 18, compared with T0, after being placed at 40°C for 4 weeks, the free drug in the eight pH buffer systems (F1-F8) increased by 25.8%-34.4% mol / mol. The free drug in F8 (histidine buffer system, pH 6.5) increased by 25.8% mol / mol, which was less than that in the other pH buffer systems (free drug increased by 29.2%-34.4% mol / mol). Therefore, F8 performed better.

[0557] Table 18. Summary of free drug results in pH buffer screening

[0558]

[0559] 3.4.10 Drug-drug conjugation ratio DAR

[0560] As shown in Table 19, compared with T0, after being placed at 40°C for 4 weeks, the DAR values ​​of the eight pH buffer systems (F1-F8) decreased by 1.84-2.95. The DAR value of F8 (histidine buffer system, pH 6.5) decreased by 1.84, which was less than that of the other pH buffer systems (DAR value decreased by 2.19-2.95). Therefore, F8 performed better.

[0561] Table 19. Summary of DAR values ​​in pH buffer screening

[0562]

[0563] 3.4.11 Summary

[0564] Based on the stability results under DSC and 40°C conditions, the F8 formulation (20 mM histidine buffer, pH 6.5) sample had a higher Tm Onset value and showed a better appearance under stability conditions, with relatively high size exclusion chromatography purity, high charge isomerization stability, smaller drug coupling ratio reduction and lower free drug. Therefore, the F8 formulation (20 mM histidine buffer, pH 6.5) was finally selected for the next round of excipient screening.

[0565] 4. Excipient screening experiment

[0566] 4.1 Experimental Purpose

[0567] The purpose of this study was to screen the optimal excipients for IEX019-02 at a concentration of 40 mg / mL in a histidine buffer system.

[0568] 4.2 Experimental plan

[0569] The formulation design and sampling plan for the excipient screening experiment are shown in Tables 20 and 21. The lyophilized samples of the three groups of formulations of four excipients were incubated under high temperature conditions for 12 weeks, and the liquid samples were incubated under high temperature conditions (40°C and 25°C for 4 weeks), freeze-thaw (5 cycles from room temperature to -70°C) and shaking conditions (150 rpm, shaking at 25°C for 3 days). The optimal excipient was selected based on the stability of the three groups of formulations. The test items included appearance, pH value, ADC molecular concentration, SE-HPLC, iCIEF, Caliper-R / NR, antibody drug conjugate ratio, free drug, collapse temperature Tc, glass transition temperature Tg', DSC, insoluble particles (HIAC method), freeze-dried sample moisture and binding activity (ELISA), etc.

[0570] Table 20. Excipient screening experiment

[0571]

[0572] Table 21. Excipient Screening Sampling Plan

[0573]

[0574] 4.3 Sample preparation

[0575] According to the composition of the stock solution and the 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 the IEX019-02 stock solution was ultrafiltered and exchanged to 20 mM histidine solution, 8.8% dihydrate trehalose (w / v), pH 6.5 solution and 20 mM histidine buffer, 2% sucrose (w / v), 4% mannitol (w / v), pH 6.5 solution, at which time the IEX019-02 concentration was higher than 40 mg / ml. Then, according to Table 20, the corresponding ultrafiltration buffer was added to dilute IEX019-02 to 40 mg / ml, and then 5% polysorbate 80 was added to 0.02% (w / v) to obtain Fe01-Fe03. After preparation, filter each prescription sample with a 0.22μm PES filter in a biosafety cabinet and fill into 20mL and 6mL vials, with a filling volume of 5mL / vial and 3mL / vial. Immediately after filling, half-stopper the 20mL vial and freeze-dry according to the procedure in Table 22, and stopper and cap the 6mL vial. Place the liquid samples under different influencing factors according to Table 21, and place at least 1 bottle of each prescription at each sampling point.

[0576] Table 22. Excipient screening lyophilization procedure

[0577]

[0578]

[0579] 4.4 Results and Discussion

[0580] The results of the excipient screening experiment are summarized in Tables 23-35.

[0581] 4.4.1 Tc, Tg' and Tm values ​​of samples

[0582] As shown in Table 23, the Tc of the three prescriptions (Fe01-Fe03) are all higher than -21℃, Fe01 and Fe02 are relatively close, Fe03 is higher, Tc is -9.2℃. Tg' is quite different, which are -28.3℃, -26.6℃ and -32.8℃ respectively. The Tm onset of the three prescriptions (Fe01-Fe03) are all higher than 67.0℃, and the Tm value is higher than 79℃, indicating that the three prescriptions have good stability.

[0583] Table 23. Summary of Tc, Tg' and Tm1 values ​​of samples

[0584]

[0585] 4.4.2 Reconstitution time and moisture results

[0586] As shown in Table 24, the reconstitution time of freeze-dried samples of the three formulations (Fe01-Fe03) was quite different, with Fe01 being relatively short. The water content of the three formulations did not change significantly during the stability process, all being less than 0.6%.

[0587] Table 24. Summary of results of reconstitution time and freeze-drying moisture content of excipient screening

[0588]

[0589] 4.4.3 Appearance

[0590] As shown in Table 25, at T0, the three formulations (Fe01-Fe03) all showed light yellow, slightly opalescent liquids with no visible particles. After shaking for 3 days, freezing and thawing 5 times, and placing at 25°C for 4 weeks, the three formulations (Fe01-Fe03) had basically no visible particles or no visible particles. However, visible particles appeared after placing at 40°C for 2 weeks and 4 weeks. The freeze-dried samples were all light yellow powder cakes before reconstitution, and basically no visible particles after reconstitution.

[0591] Table 25. Summary of appearance results of excipient screening studies

[0592]

[0593] Notes: SY: light yellow, SO: slightly opalescent liquid, FP: no visible particles, EFP: almost no visible particles (≤3), NEFP: visible particles (>3); FT-3C: freeze-thaw 3 times; FT-5C: freeze-thaw 5 times; A-1D: shake for one day; A-3D: shake for three days

[0594] 4.4.4 ADC Molecular Concentration

[0595] As shown in Table 26, compared with T0, the ADC molecular concentrations of the three formulations (Fe01-Fe03) did not change significantly after the liquid samples were placed at 40°C or 25°C for 4 weeks, 5 freeze-thaw cycles, or 25°C shaking for 3 days. The ADC molecular concentrations of the freeze-dried samples did not change significantly after being placed at 40°C for 12 weeks.

[0596] Table 26. Summary of concentration results in excipient screening studies

[0597]

[0598] 4.4.5 pH value

[0599] As shown in Table 27, the pH of the three formulations (Fe01-Fe03) did not change significantly compared with T0 after the liquid samples were placed at 40°C or 25°C for 4 weeks, 5 freeze-thaw cycles, or 25°C shaking for 3 days. The lyophilized samples were placed at 40°C for 12 weeks, and the pH did not change significantly.

[0600] Table 27. Summary of pH results in excipient screening studies

[0601]

[0602] 4.4.6 Insoluble particles (HIAC)

[0603] As shown in Table 28, compared with T0, the insoluble particles of the liquid samples of the three prescriptions (Fe01-Fe03) did not change significantly after 5 cycles of freeze-thaw. However, after shaking at 25°C for 3 days, the insoluble particles of Fe03 increased significantly, and the other samples (Fe01-Fe02) did not change significantly. The three prescriptions (Fe01-Fe03) were placed at 40°C for 2 weeks, and the particles increased significantly. The insoluble particles of the samples of the three prescriptions (Fe01-Fe03) increased slightly after freeze-drying. After freeze-drying, the samples were placed at 40°C for 2 weeks, 4 weeks and 12 weeks, and there was no significant change in the insoluble particles of Fe01, while the insoluble particles of Fe02 and Fe03 increased significantly.

[0604] Table 28. Summary of HIAC results in excipient screening studies

[0605]

[0606] 4.4.7 Size Exclusion Chromatography Purity SE-HPLC

[0607] As shown in Table 29, compared with T0, after 5 cycles of freeze-thaw or shaking at 25°C for 3 days, the SE-HPLC monomers of the liquid samples of the three prescriptions (Fe01-Fe03) did not change significantly. However, after being placed at 40°C or 25°C for 4 weeks, the SE-HPLC purity decreased significantly. There was no significant change in the SE-HPLC purity of the three prescription samples after freeze-drying. After the freeze-dried samples were placed at 40°C for 2 weeks, 4 weeks and 12 weeks, the SE-HPLC purity of Fe01, Fe02 and Fe03 decreased by 1.3%, 2.0% and 5.4%, respectively.

[0608] Table 29. Summary of SE-HPLC results in excipient screening studies

[0609]

[0610] 4.4.8 Charge isomerization iCIEF

[0611] As shown in Table 30, compared with T0, no significant changes were observed in the main iCIEF peaks of the three formulations after being placed at 25°C for 4 weeks, 5 freeze-thaw cycles, or shaking at 25°C for 3 days. After being placed at 40°C for 4 weeks, the main iCIEF peaks of the three formulations (Fe01-Fe03) decreased by 9.8%-11.8%. No significant changes were observed in the main iCIEF peaks of the three formulations (Fe01-Fe03) after freeze-drying. The freeze-dried samples were placed at 40°C for 2 weeks, 4 weeks, and 12 weeks. After 12 weeks, the main iCIEF peaks of the three formulations (Fe01-Fe03) decreased by 2.0%-3.1%.

[0612] Table 30. Summary of iCIEF results in excipient screening studies

[0613]

[0614] 4.4.9 Reduction Capillary Electrophoresis Purity Caliper-R

[0615] As shown in Table 31, compared with T0, the purity of Caliper-R in the three formulations (Fe01-Fe03) did not change significantly after 4 weeks at 25°C, 5 freeze-thaw cycles, and 3 days of shaking at 25°C. However, the purity of Caliper-R decreased slightly after the liquid sample was placed at 40°C for 4 weeks and the freeze-dried sample was placed at 40°C for 12 weeks.

[0616] Table 31. Summary of Caliper-R results from excipient screening studies

[0617]

[0618] 4.4.10 Non-reducing capillary electrophoresis purity Caliper-NR

[0619] As shown in Table 32, compared with T0, the purity of Caliper-NR of the three formulations (Fe01-Fe03) did not change significantly after the liquid samples were placed at 25°C for 4 weeks, frozen and thawed for 5 cycles, or shaken at 25°C for 3 days. After being placed at 40°C for 4 weeks, the purity of Caliper-NR of the three formulations (Fe01-Fe03) decreased by 5.3%-5.5%. After the freeze-dried samples were placed at 40°C for 12 weeks, the purity of Caliper-NR of the three formulations (Fe01-Fe03) decreased by 0.9%-2.9%.

[0620] Table 32. Summary of Caliper-NR results from excipient screening studies

[0621]

[0622] 4.4.11 Free drug

[0623] As shown in Table 33, compared with T0, the free drugs of the three prescriptions (Fe01-Fe03) did not change significantly after the liquid samples were placed at 25°C for 2 weeks, 5 freeze-thaw cycles, or 25°C shaking for 3 days. However, the free drugs increased by 1.4%-4.5% after being placed at 25°C for 4 weeks, and by 26.3%-33.7% after being placed at 40°C for 2 weeks, or by 41.6%-50.1% after being placed at 40°C for 4 weeks and 12 weeks. The free drugs of the three prescriptions (Fe01-Fe03) did not change significantly after 4 weeks, and the free drugs of the three prescriptions (Fe01-Fe03) increased by 0.9%-4.2% after 12 weeks.

[0624] Table 33. Summary of free drug results in excipient screening studies

[0625]

[0626] 4.4.12 Drug-drug conjugation ratio DAR

[0627] As shown in Table 34, compared with T0, the DAR values ​​of the three prescriptions (Fe01-Fe03) did not change significantly after the liquid samples were placed at 25°C for 4 weeks, 5 freeze-thaw cycles, or 25°C shaking for 3 days. However, the DAR values ​​decreased by 0.95-1.12 after being placed at 40°C for 2 weeks, and the DAR values ​​of the three prescriptions (Fe01-Fe03) decreased by 1.51-1.74 after 4 weeks. The freeze-dried samples were placed at 40°C for 2 weeks, 4 weeks, and 12 weeks. There was no significant change in the DAR value of 4 weeks, and the DAR values ​​of the three prescriptions (Fe01-Fe03) decreased by 0.41-0.56 after 12 weeks.

[0628] Table 34. Summary of DAR results in excipient screening studies

[0629]

[0630] 4.4.13 Binding activity

[0631] As shown in Table 35, the binding activity of the samples was tested at 2 weeks and 4 weeks. Compared with T0, there was no significant change in the activity of the liquid samples placed at 25°C for 2 weeks, lyophilized at T0, and placed at 40°C for 2 weeks and 4 weeks after lyophilization.

[0632] Table 35. Summary of excipient screening activity results

[0633]

[0634] Note: * indicates the activity test value. T0, 25℃-2W, Lyo-T0, Lyo-40℃-2W are IC50 values.

[0635] 4.4.14 Summary

[0636] Based on the overall results and the above analysis, Fe01 (20 mM histidine / histidine hydrochloride, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5) was more stable than the other formulations, so Fe01 was selected as the formulation.

[0637] 5. Prescription confirmation

[0638] 5.1 Experimental Purpose

[0639] The freeze-dried samples were prepared using the formulation selected in the excipient screening stage, and the final formulation and freeze-drying process were confirmed through long-term (2-8°C), accelerated (25±2°C) and high-temperature (40±2°C) stability experiments on the freeze-dried samples.

[0640] 5.2 Experimental plan

[0641] The stability of the lyophilized preparation was investigated on the DP prescription (finished product prescription) obtained in the excipient screening through long-term (2-8°C), accelerated (25±2°C) and high temperature (40±2°C) stability experiments to confirm the final prescription. The stability scheme is shown in Table 36.

[0642] Table 36. DP prescription confirmation scheme

[0643]

[0644] 5.3 Sample preparation

[0645] The 20 mg / mL stock solution was dispensed into 20 mL vials at 5.6 mL / bottle. The 40 mg / mL stock solution was dispensed into 20 mL vials at 5.6 mL / bottle, and then freeze-dried after half-stoppering. The freeze-drying process is shown in Table 37. The freeze-dried DP was placed under different investigation conditions (5°C long-term experiment, 25°C accelerated experiment, and 40°C high temperature experiment) according to Table 36 and sampled and tested at the specified time. Three bottles of DP were placed at each time point.

[0646] Table 37. DP freeze-drying process parameters

[0647]

[0648] 5.4 Results and Discussion

[0649] The results of stability tests under long-term, accelerated and high temperature conditions are summarized in Table 38.

[0650] The results showed that after 12 weeks of observation at 2-8°C, the appearance before reconstitution and the appearance after reconstitution of IEX019-02 samples with a concentration of 20 mg / mL and 40 mg / mL had no significant change compared with that before reconstitution at T0, and there was no significant change in pH value, ADC molecular concentration, reconstitution time, water content, antibody coupling rate, molecular exclusion chromatography purity, non-reducing capillary electrophoresis purity, reducing capillary electrophoresis purity, insoluble particles, charge isomerism and free drug, indicating that the samples have good stability at 2-8°C. However, the reconstitution time of the sample with a concentration of 20 mg / mL of IEX019-02 was shorter than that of the sample with a concentration of 40 mg / mL.

[0651] After the IEX019-02 sample with a concentration of 20 mg / mL was examined under accelerated conditions at 25°C for 12 weeks, there was no significant change in appearance before and after reconstitution compared with T0; there was no significant change in pH value, protein concentration, reconstitution time, moisture, antibody coupling rate, molecular exclusion chromatography purity, non-reducing capillary electrophoresis purity, reducing capillary electrophoresis purity, insoluble particles, charge isomerism and free drug.

[0652] After 4 weeks of observation at 40℃, the appearance of samples with IEX019-02 concentrations of 20mg / mL and 40mg / mL did not change significantly compared with T0 before and after reconstitution; pH value, protein concentration, water content, antibody coupling rate, molecular exclusion chromatography purity, non-reducing capillary electrophoresis purity, reducing capillary electrophoresis purity, insoluble particles, charge isomerism and free drugs did not change significantly. However, the reconstitution time of samples with IEX019-02 concentration of 20mg / mL was shorter than that of samples with ADC molecular concentration of 40mg / mL.

[0653] Table 38. Summary of DP prescription confirmation data

[0654]

[0655]

[0656]

[0657] Remarks: C: colorless, SY: light yellow, SO: slightly opalescent liquid, FP: no visible particles, EP: almost no visible particles (≤3).

[0658] In summary, the formulation obtained by screening showed good stability under long-term storage conditions (2-8°C) for 12 weeks, and the key quality attributes shown by the accelerated and high temperature stability tests did not change significantly compared with T0. Therefore, it was confirmed that the formulation formula of "20 mg / ml ADC, 20 mM histidine buffer, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5" was appropriate.

[0659] Sequence information:

[0660]

[0661]

Claims

1. A pharmaceutical preparation 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; The pH of the formulation is about 5.5-7.5, preferably about 6.0-6.5, more preferably about 6.

5.

2. The pharmaceutical preparation according to claim 1, wherein The antibody-drug conjugate targeting Claudin18.2 is selected from: wherein Ab is an antibody or an antigen-binding fragment thereof targeting Claudin18.2; Where q represents the average DAR, In IEX019-02, IEX019-04, and IEX019-05, q is 2-5, for example, 3-5 or 3.5-4.5, and in IEX019-03, q is 5-11, for example, 7-9 or 7.5-8.

5.

3. The pharmaceutical preparation according to claim 1, wherein the antibody-drug conjugate targeting Claudin18.2 is selected from: wherein Ab is an antibody or an antigen-binding fragment thereof targeting Claudin18.2, and In Formula IA, y is an integer of 1 or 2, In Formula IB, IC or ID, y is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

4. The pharmaceutical preparation according to claim 2 or 3, wherein The antibody or antigen-binding fragment thereof targeting Claudin18.2 comprises HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 1, 2 and 3, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 6, 7 and 8, respectively; Preferably, the antibody or antigen-binding fragment thereof targeting Claudin18.2 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region (i) comprises or consists of the amino acid sequence of SEQ ID NO: 4; or (ii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid of SEQ ID NO:4; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, preferably, the amino acid changes do not occur in the CDR regions; and / or Light chain variable region (i) comprises or consists of the amino acid sequence of SEQ ID NO: 9; or (ii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid of SEQ ID NO:9; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 9, preferably, the amino acid changes do not occur in the CDR regions.

5. The pharmaceutical preparation according to any one of claims 2 to 4, wherein the antibody or antigen-binding fragment thereof targeting Claudin18.2 comprises a heavy chain and a light chain, wherein the heavy chain (i) comprises or consists of the amino acid sequence of SEQ ID NO: 11; or (ii) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acids of SEQ ID NO: 11; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 11; and / or Light chain (i) comprises or consists of the amino acid sequence of SEQ ID NO: 12; or (ii) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acids of SEQ ID NO: 12; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:

12.

6. The pharmaceutical preparation according to any one of claims 1 to 5, wherein The pharmaceutical preparation contains about 1-200 mg / mL, preferably 15-60 mg / mL, more preferably about 20-40 mg / mL of the antibody-drug conjugate targeting Claudin18.2 or a pharmaceutically acceptable salt or solvate thereof.

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

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

9. The pharmaceutical preparation according to any one of claims 1 to 8, wherein The pharmaceutical preparation comprises about 0.01-5 mg / mL, preferably about 0.05-1 mg / mL, and more preferably about 0.1-0.3 mg / mL of a surfactant.

10. The pharmaceutical preparation according to any one of claims 1 to 9, wherein The buffer is selected from one or more of histidine, citric acid, succinic acid, glutamic acid, phosphoric acid, acetic acid or their salts; Preferably, the buffer is a combination of histidine and histidine hydrochloride or histidine.

11. The pharmaceutical preparation according to any one of claims 1 to 10, wherein The stabilizer is selected from one or more of sugars and polyols; Preferably, the sugar 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.

12. The pharmaceutical preparation according to any one of claims 1 to 11, wherein The surfactant is selected from alkyl poly(ethylene oxide), polysorbate, Pluronic or a combination thereof; Preferably, it is selected from polysorbate 20, polysorbate 80, polysorbate 60, or polysorbate 40.

13. The pharmaceutical preparation according to any one of claims 1 to 12, wherein The pharmaceutical preparation contains: (i) about 20-40 mg / ml of a Claudin18.2-targeted antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof; (ii) about 20 mM buffer; (iii) about 60-90 mg / mL of a stabilizer; and (iv) about 0.1-0.3 mg / mL surfactant.

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

15. The pharmaceutical preparation according to any one of claims 1 to 14, wherein The pharmaceutical preparation contains: (i) about 20-40 mg / mL of a Claudin18.2-targeted antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof; (ii) about 20 mM buffer; the buffer is a combination of histidine and histidine hydrochloride or histidine; (iii) about 60-90 mg / mL of a stabilizer; the stabilizer is sucrose; trehalose; or a combination of sucrose and mannitol; and (iv) about 0.1-0.3 mg / mL polysorbate 80; The pH of the formulation is about 6.0-6.5, preferably about 6.

5.

16. The pharmaceutical preparation according to any one of claims 1 to 14, wherein The pharmaceutical preparation contains: (i) about 20 mg / mL of a Claudin18.2-targeted antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof; (ii) about 20 mM buffer; the buffer is a combination of histidine and histidine hydrochloride or histidine; (iii) about 8% (w / v) stabilizer; the stabilizer is sucrose; trehalose; or a combination of sucrose and mannitol; and (iv) about 0.02% (w / v) polysorbate 80; wherein the pH of the formulation is about 6.

5.

17. A lyophilized pharmaceutical preparation containing an antibody-drug conjugate, wherein the lyophilized pharmaceutical preparation is reconstituted to form the pharmaceutical preparation according to any one of claims 1 to 16.

18. The lyophilized preparation containing an antibody-drug conjugate according to claim 17, wherein: The lyophilized pharmaceutical preparation 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, comprising (i) about 1%-60% (w / w), preferably about 15%-40% (w / w), of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof; (ii) about 0.5%-10% (w / w), preferably about 2%-5% (w / w), of a buffer; (iii) about 20% to 90% (w / w), preferably about 50% to 85% (w / w), of a stabilizer; and / or (iv) about 0.05% to 2% (w / w), preferably about 0.1% to 0.5% (w / w), of a surfactant; The antibody-drug conjugate, buffer, stabilizer and / or surfactant are as defined in claims 1-16.

20. A liquid pharmaceutical preparation containing an antibody-drug conjugate, which is obtained by reconstituting the lyophilized pharmaceutical preparation according to any one of claims 17 to 19 with a solvent, and has a pH of about 5.5-7.5, preferably about 6.0-6.5, and more preferably 6.

5.

21. The liquid pharmaceutical preparation containing an antibody-drug conjugate according to claim 20, wherein: The liquid pharmaceutical preparation is for administration by injection or infusion, for example, for intravenous injection, subcutaneous injection or intramuscular injection.

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

23. A prefilled syringe comprising the pharmaceutical preparation of any one of claims 1 to 16, or the lyophilized pharmaceutical preparation containing an antibody-drug conjugate of any one of claims 17 to 19, or the liquid pharmaceutical preparation containing an antibody-drug conjugate of claim 20 or 21, for intravenous injection, subcutaneous injection or intramuscular injection.

24. Use of the pharmaceutical preparation according to any one of claims 1 to 16, or the lyophilized pharmaceutical preparation containing an antibody-drug conjugate according to any one of claims 17 to 19, or the liquid pharmaceutical preparation containing an antibody-drug conjugate according to claim 20 or 21 in the preparation of a drug, preferably, the drug is used for preventing or treating tumors. More preferably, the tumor is a cancer, such as an epithelial cancer or a cancer of the digestive tract, such as gastric cancer, gastroesophageal junction cancer, pancreatic cancer, colorectal cancer or colon cancer.

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

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

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