Pharmaceutical composition comprising Anti-c-met antibody-drug conjugate and use thereof

Abstract

The present disclosure relates to a pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate and use thereof. In particular, the present disclosure relates to a pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and a buffer.

Description

A pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate and its use therein.

[0001] This disclosure claims priority to the following patent application: Chinese patent application filed on December 23, 2024, application number CN202411900985.3, entitled “A pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate and its use thereof”; the entire contents of the aforementioned patent application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of pharmaceutical formulations, and specifically relates to a pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate and its use therein. Background Technology

[0003] Recent studies in molecular biology and tumor pharmacology have shown that tyrosine kinase (PTK)-related cell signaling pathways play a crucial role in tumor formation and development, with over 50% of proto-oncogenes and oncogene products exhibiting tyrosine kinase activity. The c-Met proto-oncogene belongs to the Ron subfamily of the PTK family, and its encoded c-Met protein is a high-affinity receptor for hepatocyte growth factor / scatter factor (HGF / SF). The HGF / c-Met signaling pathway is closely related to angiogenesis and tumor growth; its sustained activation is a significant cause of tissue carcinogenesis or hyperproliferation of cancer cells. Inhibiting this pathway has become a novel approach for targeted cancer therapy.

[0004] The c-Met proto-oncogene is located on the long arm of human chromosome 7 (7q31), exceeding 120 kb in size, and encodes a c-Met protein precursor with a molecular weight of approximately 150 kDa. Local glycosylation generates a 170 kDa glycoprotein, which is further cleaved into an α subunit (50 kDa) and a β subunit (140 kDa), linked by disulfide bonds to form the mature c-Met protein receptor. This heterodimer contains two chains: the β chain has an extracellular region, a transmembrane region (also called a membrane extension segment), and an intracellular region (containing an intracellular tyrosine kinase binding site). The α chain has only an extracellular portion, but it is highly glycosylated and attached to the β chain via disulfide bonds. The extracellular regions of both subunits are the recognition sites for their respective ligands, while the intracellular regions possess tyrosine kinase activity.

[0005] There are three mechanisms of c-Met activation: HGF-dependent activation, HGF-independent activation, and activation via other membrane pathways, such as CD44, adhesins, and RON signaling pathways on the hyaluronic acid membrane surface. The most common is the HGF-dependent activation mechanism. The N-terminus of HGF binds to c-Met, promoting dimerization and autophosphorylation of Tyr1234 and Tyr1235 on the β-chain. Phosphorylation of Tyr1349 and Tyr1356 near the C-terminus generates binding sites for multiple adaptor proteins. These adaptor proteins induce the activation of downstream signaling mediated by Pi3K / Akt, Ras / MAPK, c-Src, and STAT3 / 5, triggering various cellular responses, such as cell survival and activity (closely related to the Pi3K / Akt pathway), tumor metastasis, and cell proliferation (mainly mediated by Ras / MAPK). In addition, c-Met cross-talks with other membrane receptors, and it is now known that such cross-talks can promote tumor formation and metastasis. Since c-Met is the intersection of many pathways that lead to tumor formation and metastasis, it is relatively easy to simultaneously interfere with many pathways by targeting c-Met. Therefore, c-Met has become a promising target for anti-tumor generation and metastasis therapy.

[0006] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to tumor cell-specific or highly expressed antigens and the high efficiency of cytotoxins, while avoiding toxic side effects on normal cells. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can precisely bind to tumor cells and reduce the impact on normal cells.

[0007] Antibody-drug conjugates (ADCs) are an important class of biopharmaceuticals. Due to their large molecular weight and complex structure, they are susceptible to degradation and polymerization caused by physical or chemical factors during production, storage, and use, leading to reduced activity or even inactivation. Furthermore, ADCs become highly unstable after conjugating small molecules to anti-c-Met antibody molecules. Therefore, selecting suitable buffer systems and stabilizers to form stable formulations is crucial for ensuring good stability of anti-c-Met ADCs during production, storage, and use. Consequently, for anti-c-Met ADCs, there is a need to develop relatively stable pharmaceutical compositions containing anti-c-Met ADCs. Summary of the Invention

[0008] This disclosure provides a pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate. It also provides a method for preparing the pharmaceutical composition, and methods for treating or preventing diseases, or related pharmaceutical uses.

[0009] Pharmaceutical Composition

[0010] This disclosure provides a pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate and a buffer.

[0011] In some embodiments, the buffer is selected from one or more of acetate buffers, succinate buffers, and histidine buffers. In some specific embodiments, the buffer is selected from one or more of acetate-sodium acetate, succinate-sodium succinate, and histidine-histidine hydrochloride. "One or more" in this disclosure includes one, two, or more.

[0012] In some embodiments, the concentration of the buffer is from about 1 mM to about 100 mM, for example, from about 1 mM to about 50 mM, from about 1 mM to about 55 mM, from about 1 mM to about 60 mM, from about 1 mM to about 70 mM, from about 1 mM to about 80 mM, from about 5 mM to about 100 mM, from about 5 mM to about 80 mM, from about 5 mM to about 70 mM, from about 5 mM to about 50 mM, from about 5 mM to about 45 mM, from about 5 mM to about 40 mM, from about 5 mM to about 35 mM, from about 5 mM to about 30 mM, from about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 35 mM, about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 15 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 35 mM, about 15 mM to about 50 mM, about 20 mM to about 35 mM, about 1 mM to about 10 mM, about 1 mM to about 20 mM, or any range between these values. In some embodiments, the concentration of the buffer is about 2 mM to about 50 mM. In some embodiments, the concentration of the buffer is about 5 mM to about 30 mM.

[0013] In some embodiments, the concentration of the buffer is about 1 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 22 mM, about 25 mM, about 26 mM, about 28 mM, about 30 mM, about 35 mM. mM, approximately 40mM, approximately 43mM, approximately 45mM, approximately 48mM, approximately 50mM, approximately 55mM, approximately 57mM, approximately 58mM, approximately 60mM, approximately 62mM, approximately 65mM, approximately 68mM, approximately 70mM, approximately 72mM, approximately 75mM, approximately 78mM, approximately 80mM, approximately 82mM, approximately 85mM, approximately 88mM, approximately 90mM, approximately 92mM, approximately 95mM, approximately 100mM.

[0014] In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 3.0 to about 8.0, for example, about 3.5 to about 7.0, about 4.0 to about 7.0, about 4.0 to about 6.5, about 4.0 to about 6.0, about 4.0 to about 5.5, about 4.0 to about 5.0, about 4.0 to about 4.8, 4.0 to about 4.5, about 4.8 to about 5.0, about 5.0 to about 5.5, or any range between these values. In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 4.0 to about 7.0.

[0015] In some embodiments, the buffer or the pharmaceutical composition has a pH value of about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.7, about 6.8, about 6.9, or about 7.0.

[0016] Typically, the pH of a pharmaceutical composition obtained by replacing a buffer is almost identical to the pH of the buffer. However, it is known to those skilled in the art that pH drift may sometimes occur during pharmaceutical formulation, but the pH drift of the pharmaceutical formulation is generally small (e.g., within ±0.8). In some embodiments, the pH drift of the pharmaceutical formulation is within ±0.5.

[0017] In some embodiments, the concentration of the anti-c-Met antibody-drug conjugate is from about 0.01 mg / mL to about 200 mg / mL, for example, from about 0.1 mg / mL to about 200 mg / mL, from about 0.5 mg / mL to about 200 mg / mL, from about 1 mg / mL to about 200 mg / mL, from about 1 mg / mL to about 180 mg / mL, from about 1 mg / mL to about 160 mg / mL, from about 1 mg / mL to about 150 mg / mL, from about 1 mg / mL to about 120 mg / mL, from about 1 mg / mL to about 110 mg / mL, from about 1 mg / mL to about 100 mg / mL, from about 1 mg / mL to about 80 mg / mL, from about 1 mg / mL to about 60 mg / mL, or any range between these values.

[0018] In some embodiments, the concentration of the anti-c-Met antibody-drug conjugate is about 0.01 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, and about... 60 mg / mL, approximately 61 mg / mL, approximately 62 mg / mL, approximately 63 mg / mL, approximately 64 mg / mL, approximately 65 mg / mL, approximately 66 mg / mL, approximately 67 mg / mL, approximately 68 mg / mL, approximately 69 mg / mL, approximately 70 mg / mL, approximately 71 mg / mL, approximately 72 mg / mL, approximately 73 mg / mL, approximately 74 mg / mL, approximately 75 mg / mL, approximately 76 mg / mL, approximately 77 mg / mL, approximately 78 mg / mL, approximately 79 mg / mL, approximately 80 mg / mL, approximately 81 mg / mL, approximately 82 mg / mL, approximately 83 mg / mL, approximately 84 mg / mL, approximately 85 mg / mL, approximately 86 mg / mL, approximately 87 mg / mL Approximately 88 mg / mL, approximately 89 mg / mL, approximately 90 mg / mL, approximately 91 mg / mL, approximately 92 mg / mL, approximately 93 mg / mL, approximately 94 mg / mL, approximately 95 mg / mL, approximately 96 mg / mL, approximately 97 mg / mL, approximately 98 mg / mL, approximately 99 mg / mL, approximately 100 mg / mL, approximately 101 mg / mL, approximately 102 mg / mL, approximately 103 mg / mL, approximately 104 mg / mL, approximately 105 mg / mL, approximately 106 mg / mL, approximately 107 mg / mL, approximately 108 mg / mL, approximately 109 mg / mL, approximately 110 mg / mL, approximately 111 mg / mL, approximately 112 mg / mL, approximately 113 mg / mL. Approximately 114 mg / mL, approximately 115 mg / mL, approximately 116 mg / mL, approximately 117 mg / mL, approximately 118 mg / mL, approximately 119 mg / mL, approximately 120 mg / mL, approximately 121 mg / mL, approximately 122 mg / mL, approximately 123 mg / mL, approximately 124 mg / mL, approximately 125 mg / mL, approximately 126 mg / mL, approximately 127 mg / mL, approximately 128 mg / mL, approximately 129 mg / mL, approximately 130 mg / mL, approximately 131 mg / mL, approximately 132 mg / mL, approximately 133 mg / mL, approximately 134 mg / mL, approximately 135 mg / mL, approximately 140 mg / mL, approximately 145 mg / mL, approximately 150 mg / mL.Approximately 155 mg / mL, approximately 160 mg / mL, approximately 165 mg / mL, approximately 170 mg / mL, approximately 175 mg / mL, approximately 180 mg / mL, approximately 185 mg / mL, approximately 190 mg / mL, approximately 195 mg / mL, approximately 200 mg / mL.

[0019] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188), polysorbates (e.g., polysorbate 20 (i.e., PS20), polysorbate 80 (i.e., PS80)), polyhydroxyalkanoates, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-saccharide, linoleyl-saccharide, succinate ... Betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocarbamate-propyl-betaine, linoleamide-propyl-betaine, myristamidopropyl-betaine, palmitoamide-propyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoamide-propyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl, sodium methyl oleate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc., or any combination thereof. In some embodiments, the surfactant is sodium dodecyl sulfate (SDS) or polysorbate. In some embodiments, the surfactant is polysorbate 80.

[0020] In some embodiments, the concentration of the surfactant is about 0.01 to 0.10% (w / v), for example about 0.01 to 0.09% (w / v), about 0.01 to 0.08% (w / v), about 0.01 to 0.07% (w / v), about 0.01 to 0.06% (w / v), about 0.01 to 0.05% (w / v), and any range between these values.

[0021] In some embodiments, the concentration of the surfactant is about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), or about 0.10% (w / v).

[0022] In some embodiments, the pharmaceutical composition comprises sugars or alcohols.

[0023] In some embodiments, the sugar or alcohol is selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, xylitol, sorbitol, mannitol, melitriose, maltotriose, mannose, stachyose, maltose, lactulose, maltitol, lactitol, and isomaltulose.

[0024] In some embodiments, the sugar is sucrose or trehalose.

[0025] In some embodiments, the concentration of the sugar or alcohol is from about 0.1% (w / v) to about 20% (w / v), for example from about 1% (w / v) to about 18% (w / v), from about 1% (w / v) to about 15% (w / v), from about 1% (w / v) to about 12% (w / v), from about 1% (w / v) to about 8% (w / v), from about 1% (w / v) to about 8.8% (w / v), from about 2% (w / v) to about 20% (w / v), from about 2 % (w / v) to about 15% (w / v), about 2% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2% (w / v) to about 8.8% (w / v), about 3% (w / v) to about 20% (w / v), about 3% (w / v) to about 15% (w / v), about 3% (w / v) to about 10% (w / v), about 3% (w / v) to about 8% (w / v), about 3% (w / v) to about 8.8% (w / v), about 4% (w / v) to about 20% (w / v), about 4% (w / v) to about 15% (w / v), about 4% (w / v) to about 10% (w / v), about 4% (w / v) to about 8% (w / v), about 4% (w / v) to about 8.8% (w / v), about 5% (w / v) to about 20% (w / v), about 5% (w / v) to about 15% (w / v), about 5% (w / v) to about 10% (w / v) ), approximately 5% (w / v) to approximately 8% (w / v), approximately 5% (w / v) to approximately 8.8% (w / v), approximately 4.4% (w / v) to approximately 20% (w / v), approximately 4.4% (w / v) to approximately 15% (w / v), approximately 4.4% (w / v) to approximately 10% (w / v), approximately 4.4% (w / v) to approximately 8% (w / v), approximately 4.4% (w / v) to approximately 8.8% (w / v), and any range between these point values. In some embodiments, the concentration of the sugar is about 1% (w / v), 1.2% (w / v), 1.4% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 4.2% (w / v), about 4.4% (w / v), about 4.8% (w / v), about 5% (w / v), about 6% (w / v), or about 7%. (w / v), approximately 7.8% (w / v), approximately 8% (w / v), approximately 8.2% (w / v), approximately 8.4% (w / v), approximately 8.8% (w / v), approximately 9% (w / v), approximately 10% (w / v), approximately 12% (w / v), approximately 14% (w / v), approximately 16% (w / v), approximately 18% (w / v), approximately 20% (w / v).

[0026] In some embodiments, the pharmaceutical composition comprises:

[0027] (A) Approximately 1–100 mg / mL of anti-c-Met antibody-drug conjugate,

[0028] Approximately 5–20 mM acetic acid-sodium acetate,

[0029] Approximately 2–10% (w / v) sucrose or trehalose,

[0030] Approximately 0.01–0.05% (w / v) polysorbate 80,

[0031] The pH of the pharmaceutical composition is 4.0–6.0;

[0032] or

[0033] (B) Approximately 1–100 mg / mL of anti-c-Met antibody-drug conjugate,

[0034] Approximately 5–20 mM succinic acid-sodium succinate,

[0035] Approximately 2-10% (w / v) sucrose,

[0036] Approximately 0.01–0.05% (w / v) polysorbate 80,

[0037] The pH of the pharmaceutical composition is 5.0–6.0;

[0038] or

[0039] (C) Approximately 1–100 mg / mL of anti-c-Met antibody-drug conjugate,

[0040] Approximately 5–20 mM histidine-histidine hydrochloride.

[0041] Approximately 2-10% (w / v) sucrose,

[0042] Approximately 0.01–0.05% (w / v) polysorbate 80,

[0043] The pH of the pharmaceutical composition is 5.5 to 6.5.

[0044] This disclosure also provides a lyophilized formulation obtained by freeze-drying a liquid formulation comprising the above-described pharmaceutical composition.

[0045] This disclosure also provides a reconstituted solution, which is prepared by reconstituted the lyophilized formulation.

[0046] In one embodiment, the reconstituted solution is obtained by reconstituted the lyophilized preparation with a solvent; in a specific embodiment, the solvent is preferably water, physiological saline, or glucose.

[0047] This disclosure also provides an article comprising a container containing the pharmaceutical composition, the lyophilized formulation, or the reconstituted solution as described.

[0048] This disclosure also provides the use of the pharmaceutical composition, the lyophilized formulation, the reconstituted solution, or the product thereof in the preparation of a medicament for the prevention or treatment of a disease;

[0049] In one implementation, the disease or condition is cancer, or a c-Met-mediated disease or condition.

[0050] In one implementation, the cancer is a cancer that expresses c-Met.

[0051] In one specific embodiment, the cancer is stomach cancer, lung cancer, intestinal cancer, or liver cancer.

[0052] Antibody-drug conjugates

[0053] This disclosure provides an antibody-drug conjugate targeting c-Met or a pharmaceutically acceptable salt thereof, having the structure shown in formula (I):

[0054] Ab-(LYD)n Formula (I)

[0055] in:

[0056] Ab is an anti-c-Met antibody, which contains a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH contains HCDR1, HCDR2 and HCDR3 in any of the amino acid sequences shown in SEQ ID NO: 10-12, and the VL contains LCDR1, LCDR2 and LCDR3 in any of the amino acid sequences shown in SEQ ID NO: 13-15.

[0057] The CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system;

[0058] -L- represents the connector unit, which is -L 1 -L 2 -L 3 -L 4 -,

[0059] L 1 It is -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, where W is selected from C. 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight-chain heteroalkyl with 1 to 8 atoms, wherein the heteroalkyl comprises 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0060] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 Integers from 1 to 20;

[0061] L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0062] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - or chemical bond, where t is an integer from 1 to 6;

[0063] R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

[0064] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

[0065] Y is selected from -O-(CR) a R b ) m -CR 1 R 2 -C(O)-、-O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CRa R b ) m -CR 1 R 2 -C(O)- or -S-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0066] R a and R b They may be the same or different, and each is independently selected from hydrogen, deuterium, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, or heterocyclic groups; or, R a and R b Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0067] R 1 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

[0068] R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

[0069] Or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0070] Or, R a and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0071] m is an integer from 0 to 4;

[0072] n is between 1 and 10, and n can be an integer or a decimal;

[0073] D represents camptothecin-type drugs.

[0074] In some implementations, D is eczema or a derivative thereof.

[0075] In some implementation schemes, L 1 Connected to Ab, L 4 Connect to Y.

[0076] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) above, wherein the anti-c-Met antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH and VL are selected from any group thereof:

[0077] (1) The VH contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1-3, and the VL contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, 5 and 6, for example, the amino acid sequence shown in SEQ ID NO: 22 is RAX1KSVSTSTYNYLH, where X1 is selected from N or D;

[0078] Table 1. CDR Sequences

[0079] (2) The VH contains HCDR1, HCDR2 and HCDR3 in any of the amino acid sequences shown in SEQ ID NO: 10-12, and the VL contains LCDR1, LCDR2 and LCDR3 in any of the amino acid sequences shown in SEQ ID NO: 13-15.

[0080] In some implementations, the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR is defined according to the Kabat numbering system.

[0081] In some embodiments, the anti-c-Met antibody comprises VH and VL, wherein any HCDR contained in the VH has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned HCDRs; and / or any LCDR contained in the VL has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned LCDRs. In some specific embodiments, the aforementioned amino acid mutations are conserved substitutions, replacements, or modifications, and / or deletions or additions that do not affect function.

[0082] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof of formula (I) as described in any of the preceding claims, wherein the anti-c-Met antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, 2 and 3 in its VH, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 7, 5 and 6 in its VL.

[0083] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof of formula (I) as described in any of the preceding claims, wherein the anti-c-Met antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, 2 and 3 in its VH, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, 5 and 6 in its VL.

[0084] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding embodiments, wherein the anti-c-Met antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0085] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof of formula (I) as described in any of the preceding claims, wherein the anti-c-Met antibody comprises a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 9. The anti-c-Met antibody is a murine antibody.

[0086] Heavy chain variable region:

[0087] Light chain variable region:

[0088] The amino acid residues of the VH / VL CDR of anti-human c-Met antibody are determined and annotated by the Kabat numbering system, and the CDR is the part in bold.

[0089] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding claims, wherein the c-Met antibody further comprises a heavy chain FR region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof in its heavy chain variable region.

[0090] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding claims, wherein the c-Met antibody has a heavy chain FR region sequence on its heavy chain variable region derived from a human heavy chain sequence, for example, human heavy chain IGHV 3-33*01; a framework sequence comprising the FR1, FR2, FR3 and FR4 regions of human heavy chain IGHV 3-33*01 or a mutated sequence thereof, for example, the mutated sequence being a reversion mutation of 0-10 amino acids.

[0091] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in any of the preceding formulas (I), wherein the c-Met antibody has a light chain FR region sequence on its light chain variable region selected from human light chain sequences, such as human light chains IGKV085 or IGKV 4-1*01, comprising the framework sequence of the FR1, FR2, FR3 and FR4 regions of human light chains IGKV085 and IGKV 4-1*01 or a mutated sequence thereof, for example, the mutated sequence being a reversion mutation of 0-10 amino acids.

[0092] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding claims, wherein the anti-c-Met antibody is a humanized antibody. In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding claims, wherein the anti-c-Met antibody comprises a heavy chain variable region sequence selected from SEQ ID NO: 10-12 and a light chain variable region sequence selected from SEQ ID NO: 13-15.

[0093] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding claims, wherein the anti-c-Met antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence selected from any one of a) to c):

[0094] a) The heavy chain variable region sequence of SEQ ID NO: 10 or a heavy chain variable region sequence having at least 90% sequence identity with it, and the light chain variable region sequence of SEQ ID NO: 13 or a light chain variable region sequence having at least 90% sequence identity with it;

[0095] b) The heavy chain variable region sequence of SEQ ID NO: 11 or a heavy chain variable region sequence having at least 90% sequence identity with it, and the light chain variable region sequence of SEQ ID NO: 14 or a light chain variable region sequence having at least 90% sequence identity with it;

[0096] Alternatively, c) the heavy chain variable region sequence of SEQ ID NO: 12 or a heavy chain variable region sequence having at least 90% sequence identity with it, and the light chain variable region sequence of SEQ ID NO: 15 or a light chain variable region sequence having at least 90% sequence identity with it.

[0097] In some embodiments, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof of formula (I) as described in any of the preceding claims, wherein the anti-c-Met antibody comprises the heavy chain variable region sequence of SEQ ID NO: 11 and the light chain variable region sequence of SEQ ID NO: 14.

[0098] Heavy chain variable region:

[0099] Ab-9

[0100] Ab-10

[0101] Ab-11

[0102] Light chain variable region:

[0103] Ab-9

[0104] Ab-10

[0105] Ab-11

[0106] In some embodiments, the anti-c-Met antibody further comprises a light chain constant region and / or a heavy chain constant region.

[0107] In some embodiments, the light chain constant region is derived from the κ light chain, the λ light chain, or a variant of any of the foregoing. For example, it is derived from the human κ light chain, the human λ light chain, or a variant of any of the foregoing. In some specific embodiments, the light chain constant region is derived from the human κ light chain or a variant thereof.

[0108] In some implementations, the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any of the aforementioned. For example, it may be derived from human IgG1, human IgG2, human IgG3, human IgG4, or a variant of any of the aforementioned.

[0109] In some embodiments, the anti-c-Met antibody further comprises an Fc region. In some specific embodiments, the Fc region is derived from the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some specific embodiments, the Fc region is the Fc region of human IgG2.

[0110] In some embodiments, the c-Met antibody, as shown in formula (I) of any of the preceding embodiments, is a humanized antibody, wherein the heavy chain constant region of the humanized antibody comprises a constant region derived from human IgG1 or a variant thereof, human IgG2 or a variant thereof, human IgG3 or a variant thereof, or human IgG4 or a variant thereof. In some embodiments, the heavy chain constant region of the humanized antibody comprises a constant region of human IgG1 or a variant thereof, or human IgG2 or a variant thereof, or human IgG4 or a variant thereof. In some embodiments, the heavy chain constant region of the humanized antibody comprises a constant region of human IgG2 or a variant thereof.

[0111] In some embodiments, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding embodiments, wherein the anti-c-Met antibody:

[0112] (1) The heavy chain contains an amino acid sequence as shown in any of SEQ ID NO: 16, 18, 20 or having at least 80% sequence identity with it; and / or, the light chain contains an amino acid sequence as shown in any of SEQ ID NO: 17, 19, 21 or having at least 80% sequence identity with it;

[0113] (2) The heavy chain contains an amino acid sequence as shown in SEQ ID NO: 16 or having at least 80% sequence identity with it; and / or, the light chain contains an amino acid sequence as shown in SEQ ID NO: 17 or having at least 80% sequence identity with it;

[0114] (3) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 18 or having at least 80% sequence identity with it; and / or, the light chain comprises an amino acid sequence as shown in SEQ ID NO: 19 or having at least 80% sequence identity with it; or

[0115] (4) The heavy chain contains an amino acid sequence as shown in SEQ ID NO: 20 or having at least 80% sequence identity with it; and / or, the light chain contains an amino acid sequence as shown in SEQ ID NO: 21 or having at least 80% sequence identity with it.

[0116] In the context of this disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity.

[0117] Ab-9 humanized antibody:

[0118] Heavy chain:

[0119] Light chain:

[0120] Ab-10 humanized antibody:

[0121] Heavy chain:

[0122] Light chain:

[0123] Ab-11 humanized antibody:

[0124] Heavy chain:

[0125] Light chain:

[0126] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding claims, wherein the anti-c-Met antibody comprises a heavy chain sequence as shown in SEQ ID NO: 18 and a light chain sequence as shown in SEQ ID NO: 19.

[0127] In some embodiments, the anti-c-Met antibody encompasses an antigen-binding fragment. In some embodiments, the antigen-binding fragment includes, but is not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, and domain antibodies. For example, the antigen-binding fragment is an scFv, Fv, Fab, or Fab' fragment. In some embodiments, the anti-c-Met antibody includes monospecific antibodies and multispecific antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies). In some embodiments, the anti-c-Met antibody includes diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv, etc.

[0128] In some embodiments, the anti-c-Met antibody in the antibody-drug conjugate binds to or competes with any of the aforementioned anti-c-Met antibodies for the same c-Met epitope.

[0129] In some embodiments, the anti-c-Met antibody in the antibody-drug conjugate blocks the binding of any of the aforementioned anti-c-Met antibodies to c-Met.

[0130] In some embodiments, the binding of the anti-c-Met antibody in the antibody-drug conjugate to c-Met is blocked by any of the aforementioned anti-c-Met antibodies.

[0131] In some implementations, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (I) of any of the preceding embodiments,

[0132] in:

[0133] Y is -O-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0134] R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, and alkyl groups;

[0135] R 1 Selected from hydrogen atoms, haloalkyl groups, or C 3-6 cycloalkyl;

[0136] R 2 Selected from hydrogen atoms, haloalkyl groups and C 3-6 cycloalkyl;

[0137] Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl;

[0138] m is 0 or 1.

[0139] In some embodiments, the antibody-drug conjugate of formula (I) (Pc-LYD) as described in any of the preceding embodiments, wherein Y is selected from:

[0140] The O end of Y is connected to the connector unit L.

[0141] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt of the general formula (I) (Pc-LYD) as described in any of the preceding embodiments is wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0142] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is selected from C. 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight-chain heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl group comprises 1 to 3 heteroatoms selected from N, O and S, wherein the C 1-8. Each of the alkyl, cycloalkyl and straight-chain heteroalkyl groups may be independently and optionally further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0143] L 2 Selected from -NR 4 (CH2CH2O)pCH2CH2C(O)-、-NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)-, or chemical bonds, where p is an integer from 1 to 20;

[0144] L 3 It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl.

[0145] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - and chemical bonds, where t is an integer from 1 to 6;

[0146] R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

[0147] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

[0148] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt of the general formula (I) (Pc-LYD) as described in any of the preceding embodiments is wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0149] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR3 -WC(O)- and -C(O)-WC(O)-, where W is selected from C. 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight-chain heteroalkyl groups with 1 to 8 chain atoms, wherein the heteroalkyl group comprises 1 to 3 heteroatoms selected from N, O and S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0150] L 2 Selected from -NR 4 (CH2CH2O)pCH2CH2C(O)-、-NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)-, or chemical bonds, where p is an integer from 1 to 20;

[0151] L 3 It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0152] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - and chemical bonds, where t is an integer from 1 to 6, and non-limiting examples are 1, 2, 3, 4, 5 and 6;

[0153] R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

[0154] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

[0155] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt of the general formula (I) (Pc-LYD) as described in any of the preceding embodiments is wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0156] L 1 for s 1 The integer is from 2 to 8, with unrestricted instances being 2, 3, 4, 5, 6, 7, and 8;

[0157] L 2 It is a chemical bond;

[0158] L 3 It is a tetrapeptide residue; for example, L 3 It is a tetrapeptide residue of GGFG;

[0159] L 4 For -NR 5 (CR 6 R 7 )t-,R 5 R 6 or R 7 They may be the same or different, and each is independently a hydrogen atom or an alkyl group, with t being 1 or 2;

[0160] The L mentioned therein 1 The terminal is connected to the PC, L 4 The end is connected to Y.

[0161] In some implementations, the camptothecin-based drug, such as eczema, is used.

[0162] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof, as described in any of the preceding embodiments, is an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof:

[0163] in:

[0164] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight-chain heteroalkyl with 1 to 8 atoms, wherein the heteroalkyl comprises 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0165] L 2Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 Integers from 1 to 20;

[0166] L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0167] R 1 Selected from hydrogen atoms, halogens, cycloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups;

[0168] R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

[0169] Or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0170] R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

[0171] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

[0172] m is an integer from 0 to 4;

[0173] n is between 1 and 10, and n can be an integer or a decimal;

[0174] Ab is as defined in general formula (I).

[0175] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in formula (II) of any of the preceding embodiments, is 1 to 10, for example, 1 to 8, 2 to 8, 2 to 7, 2 to 4, 3 to 8, 3 to 7, 3 to 6, 4 to 7, or 4 to 6, and n is a decimal or an integer. In some embodiments, n is 1 to 8, and n is a decimal or an integer. In some embodiments, n is 3 to 7, and n is a decimal or an integer. In some embodiments, n is 4 to 6, and n is a decimal or an integer. In some embodiments, n is an average of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. In some implementations, n is an average of approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, 4.7, approximately 4.8, approximately 4.9, approximately 5, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, or approximately 6.

[0176] In some embodiments, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in formula (II) of any of the preceding embodiments, wherein:

[0177] The -LY- structure is as follows:

[0178] s 1 Integers between 2 and 8;

[0179] L 2 L 3 R 1 R 2 R 5 R 6 R 7 And m are as defined in the aforementioned general formula (II).

[0180] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, representing general formula (I) as described in any of the preceding embodiments, is of general formula (III)(Ab-L b The antibody-drug conjugate or its pharmaceutically acceptable salt shown in (-YD) is:

[0181] in:

[0182] s 1 Integers between 2 and 8;

[0183] Pc, R 1 R 2 R 5 R 6 R 7 m and n are as defined in general formula (II).

[0184] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt of general formula (I) as described in any of the preceding embodiments is:

[0185] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, wherein -LY- is selected from:

[0186] In some implementations, -LY- is selected from:

[0187] In some implementations, -LY- is

[0188] In some implementations, -LY- means:

[0189] In some embodiments, the antibody-drug conjugate represented by general formula (I) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, is selected from:

[0190] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, is:

[0191] In some embodiments, the antibody-drug conjugate represented by general formula (I) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, is selected from:

[0192] Where Ab and n are as defined in general formula (I).

[0193] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, is:

[0194] in:

[0195] n is 1 to 8, and n is a decimal or an integer, such as an integer or decimal of 3 to 7, or an integer or decimal of 4 to 6;

[0196] Ab is an anti-c-Met antibody, which contains a heavy chain as shown in SEQ ID NO: 18 and a light chain as shown in SEQ ID NO: 19.

[0197] Preparation method

[0198] In some embodiments, this disclosure provides a method for preparing an antibody-drug conjugate, comprising: conjugating any one of the aforementioned anti-c-Met antibodies with a drug to obtain the antibody-drug conjugate. Further, the method further includes: purifying the antibody-drug conjugate.

[0199] In some embodiments, a method for preparing an antibody-drug conjugate as shown in general formula (I) includes the following steps:

[0200] After reduction of Ab, it undergoes a coupling reaction with general formula (La-YD) to give the compound shown in general formula (II);

[0201] Wherein, Ab is the anti-c-Met antibody disclosed herein; W, L 2 L 3 R 1 R 2 R 5 To R 7 m and n are as defined in equation (I).

[0202] In some implementations, the reducing agent is TCEP.

[0203] In some implementations, the reducing agent reduces the disulfide bonds on the antibody.

[0204] the term

[0205] To facilitate understanding of this disclosure, certain techniques and scientific methods are specifically defined below. Unless otherwise expressly defined in this disclosure, all other techniques and scientific methods used in this disclosure have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0206] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as having an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of “including but not limited to.”

[0207] "Buffer" refers to a buffer that is resistant to pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0208] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include acetate-histidine, succinate-histidine, histidine-histidine hydrochloride, hydrochloride-histidine, sulfate-histidine, and other buffers. For example, a histidine-hydrochloride buffer is prepared by reacting histidine with hydrochloric acid or histidine with histidine hydrochloride.

[0209] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity. In this document, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0210] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0211] As used herein, the term "about" means a numerical value within the acceptable margin of error of a specific value as determined by one of ordinary skill in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, "about" may mean within or above 1 standard deviation. Alternatively, "about" or "substantially includes" may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to five times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "substantially includes" should be assumed to be within the acceptable margin of error of that specific value.

[0212] The pharmaceutical compositions described in this disclosure achieve a stable effect: the antibodies therein substantially retain their physical and / or chemical stability and / or biological activity after storage; preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0213] Stable drug antibody formulations are those in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for 1 month, 3 months, 6 months, or at 40°C for 1 month. Typical acceptable criteria for stability are as follows: degradation of antibody monomers typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the drug antibody formulation is colorless to yellow, clear to slightly opalescent. The concentration, pH, and osmotic pressure of the formulation exhibit variations not exceeding ±10%. Truncation typically not exceeding about 10%, preferably not exceeding about 5%, and aggregation typically not exceeding about 10%, preferably not exceeding about 5%, are observed.

[0214] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody “retains its physical stability” in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0215] If an antibody does not show significant chemical changes, then the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0216] If the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared, then the antibody "retains its biological activity" in the pharmaceutical formulation. The biological activity of an antibody can be determined, for example, by an antigen-binding assay.

[0217] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0218] The terms “about” and “approximately” mean that a numerical value is within the acceptable error range of a specific value as determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, “about” may mean within or above 1 standard deviation. Alternatively, “about” or “substantially includes” may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%. In this disclosure, each instance of a number or range of values ​​preceded by the term “about” also includes embodiments of a given number. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially includes” should be assumed to be within the acceptable error range of that specific value.

[0219] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be used to provide clear support for both meanings or either meaning.

[0220] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0221] The terms "c-Met," "c-Met polypeptide," or "c-Met receptor" refer to receptor tyrosine kinases that bind to cell growth factor (HGF). Unless otherwise specified, in this disclosure, mouse c-Met (mc-Met) or monkey c-Met (cyno-c-Met) refers to human c-Met (hc-Met). Human, mouse, and cynomolgus monkey c-Met as used in this disclosure are encoded by nucleotide or polypeptide sequences provided in GenBank, such as the human polypeptide encoded by the nucleotide sequence provided in GenBank accession number NM_000245, or the human protein or its extracellular domain encoded by the polypeptide sequence provided in GenBank accession number NP_000236. The original single-chain precursor protein is cleaved post-translational to produce α and β subunits, which are linked by disulfide bonds to form the mature receptor. Receptor tyrosine kinase c-Met participates in cellular processes including, for example, migration, invasion, and morphogenesis associated with tissue regeneration during embryonic development.

[0222] The term "c-Met-related condition" refers to any disease, symptom, or condition arising from unfavorable or absent expression, unfavorable or absent regulation, or harmful or absent activity of c-Met, or that can be regulated, treated, or cured by modulating c-Met expression or activity. For example, activation of the HGF / c-Met pathway can be expected in most cancer patients, or in patients whose disease is indeed driven by changes related to the c-Met pathway. This upregulation may be attributed to different mechanisms, such as overexpression of HGF and / or c-Met, or constitutive activation via c-Met mutations. c-Met-related conditions include, but are not limited to, proliferative diseases, including but not limited to cancers such as gastric cancer, lung cancer, colorectal cancer, and / or liver cancer.

[0223] The term "antibody" is used in the broadest sense, encompassing various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies can also refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five types of Ig can have either a κ chain or a λ chain.

[0224] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are known as the constant region (C region). The variable region includes three hypervariable regions (CDRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0225] In this disclosure, the antibody light chain may further include a light chain constant region. Exemplarily, the light chain constant region comprises a human or mouse κ, λ chain, or a variant thereof.

[0226] In this disclosure, the antibody heavy chain may further include a heavy chain constant region. Exemplarily, the heavy chain constant region includes human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof.

[0227] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human c-Met prepared in mice according to the knowledge and skills of the art. Preparation involves injecting the test subject with c-Met antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional characteristics. In one embodiment of this disclosure, the mouse c-Met antibody may further comprise a light chain constant region of a mouse κ, λ chain, or a variant thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

[0228] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes a murine-specific monoclonal antibody must first be established. Then, the variable region gene is cloned from mouse hybridoma cells, and subsequently, the constant region gene of the human antibody is cloned into it for recombinant expression.

[0229] The term "humanized antibody," also known as CDR-grafted antibody, refers to antibodies produced by grafting mouse CDR sequences into the variable region framework of human antibodies, i.e., into different types of human germline antibody framework sequences. This can overcome the strong antibody-variable antibody response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the VBase human germline sequence database and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. The human antibody variable region framework is designed and selected such that the light chain FR region sequence on the antibody light chain variable region is selected from human germline light chain sequences, such as human germline light chains IGKV085 or IGKV 4-1*01, including the FR1, FR2, FR3, and FR4 regions of human germline light chains IGKV085 and IGKV 4-1*01; and the heavy chain FR region sequence on the antibody heavy chain variable region is derived from human germline heavy chain sequences, such as human germline heavy chain IGHV 3-33*01, including the FR1, FR2, FR3, and FR4 regions of human germline heavy chain IGHV 3-33*01. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region can be subjected to minimal reversion mutations to maintain activity.

[0230] The term "antigen binding site" can refer to a discontinuous three-dimensional spatial site on the antigen that is recognized by the antibody or antigen-binding fragment disclosed herein.

[0231] The term "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or non-adjacent amino acids (which are spatially close to each other through the ternary folding of a protein). Epitopes formed from adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost after treatment with denaturing solvents. Epitopes typically exist in a unique spatial conformation, comprising at least 3-15 amino acids. Methods for determining epitopes are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0232] "Specific binding" or "selective binding" refers to the binding of an antibody to a predetermined epitope on an antigen. Typically, human c-Met or its epitopes are used as analytes, and antibodies are used as the target antibody. When measured in an instrument using surface plasmon resonance (SPR) technology, the antibody binds at a rate of approximately less than 10... -7 M or even smaller equilibrium dissociation constant (K) D It binds to a predetermined antigen or its epitope, and its affinity for binding to the predetermined antigen or its epitope is at least twice that for binding to the predetermined antigen or its epitope (or a non-specific antigen other than a closely related antigen, such as BSA).

[0233] “Affinity” refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its bound antibody (e.g., an antigen). Unless otherwise specified, as used herein, “binding affinity” refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its antibody Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The terms “kassoc” or “ka” refer to the association rate of a particular antibody-antigen interaction, while the terms “kdis” or “kd” as used herein are intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term “KD” refers to the equilibrium dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art, such as measuring surface plasmon resonance using a biosensing system, or measuring affinity in solution by solution equilibrium titration (SET). "Cross-reactivity" refers to the ability of an antibody (or fragment thereof) of this disclosure to bind to c-Met from a different species. For example, an antibody of this disclosure that binds to human c-Met may also bind to c-Met from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in a binding assay (e.g., SPR or ELISA), or by binding or functional interaction with cells physiologically expressing c-Met. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.

[0234] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two compared sequences are homologous at positions occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences. To determine the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the compared sequences, such as BLAST.

[0235] The term "internalization" refers to the transport of a portion of a cell from its exterior to its interior. The internalized portion may reside within intracellular compartments. "Internalized" or "internalized" antigens or antibodies are those capable of being transported from the exterior to the interior of a target cell. It is generally understood by those skilled in the art that the process of cell internalization typically refers to the transmembrane movement of cell surface molecules from the cell surface to the cell interior. After internalization, endosomes can be transported to lysosomes for degradation or recycled back to the cell surface. The rate of internalization of a given cell surface molecule provides a measurement of the kinetics of the molecule's movement from the cell surface across the plasma membrane to the cell interior. The internalization activity or rate of antigens and antibodies can be monitored and / or measured by a variety of techniques known in the art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305–17, 2008) and toxicity assays (Pahara J. et al., Exp Cell Res., 316:2237–50, 2010; and Mazor et al., J. Immunol. Methods, 321:41–59, 2007). Many antibody labeling techniques, dyes, and kits for antibody labeling are commercially available for quantifying and monitoring internalization (e.g., the pHrodo iFL antibody labeling method, reagents, and kits sold by Thermo Fisher Scientific).

[0236] For example, antibody-drug conjugates (ADCs) bind to tumor cell surface antigens via antibodies within the ADC, then undergo endocytosis into the endosome, and are subsequently converted from the endosome to the lysosome. In the lysosome, hydrolytic enzymes dissociate the bioactive molecules (e.g., toxins or payloads) from the ADC. These dissociated bioactive molecules enter the cytoplasm from the lysosome and kill tumor cells. Bioactive molecules that escape after killing tumor cells can further kill surrounding tumor cells that do not express or express low levels of the antigen (the so-called by-stander effect).

[0237] A "mutant" or "variant" is a polypeptide that contains at least one amino acid mutation (such as substitution, deletion, or insertion) compared to the amino acid sequence of the "parent," as long as the variant can still bind to the antigen or fragments thereof. Amino acid mutations (also known as amino acid modifications) include, for example, the deletion and / or insertion and / or substitution of residues in the amino acid sequence of a binding molecule (e.g., an antibody). Any combination of deletions, insertions, and substitutions can be introduced into the "parent" amino acid sequence to obtain the final variant. Amino acid mutations also include alterations to the post-translational processes of the binding molecule, such as changing the number or position of glycosylation sites. For example, depending on the length of the CDR and FR themselves, 1, 2, 3, 4, 5, or 6 amino acids can be inserted into or deleted from each CDR, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids can be inserted into or deleted from each FR. Furthermore, amino acid substitutions can be introduced into the CDR, VH, or FR regions of the heavy and / or light chains. For example, conserved substitutions.

[0238] "Conservative substitution" refers to replacing an amino acid residue with another amino acid residue that has similar properties to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Additionally, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when amino acid residues in the group exhibiting similar properties as described above are substituted, it will not exhibit a specific change in properties.

[0239] The term "camptothecin class of drugs" refers to camptothecin and its derivatives, which are cytotoxic, and are not limited to 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, ixotecan, irinotecan or 9-nitro-10-hydroxycamptothecin and its derivatives or pharmaceutically acceptable salts.

[0240] The term "antibody-drug conjugate" (ADC) refers to an antibody linked to a drug via a linker (or connecting unit). In some embodiments, within this disclosure, "antibody-drug conjugate" refers to the linking of an anti-C-met antibody to a toxic drug via a connecting unit.

[0241] The term "drug" refers to a cytotoxic drug, denoted by D, which is a chemical molecule that strongly disrupts the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. This term also includes toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and ribolysins, preferably toxic drugs.

[0242] The term "linker unit," "connector fragment," or "connector cell" refers to a chemical structural fragment or bond that is connected to an antibody at one end and to a drug at the other end. It can also be connected to other linkers before being linked to the drug. In a non-limiting scheme, it is represented by L and L1 to L4, where L1 is connected to the antibody, and L4 is connected to structural unit Y before being linked to the drug (D).

[0243] The term "drug loading" refers to the average amount of cytotoxic drug loaded onto each ligand (anti-c-Met antibody) in an ADC, and can also be expressed as the ratio of drug amount to antibody amount. Drug loading can range from 1 to 20 cytotoxic drugs (D) linked to each antibody (Pc), for example, 1 to 10 cytotoxic drugs (D). In embodiments of this disclosure, drug loading is expressed as n, exemplarily 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values; therefore, drug loading can be an integer or a decimal. The average amount of drug per ADC molecule after the coupling reaction can be characterized using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, CE-SDS (monoclonal antibody molecular size variant assay), and HPLC.

[0244] The present invention discloses a method for determining the molecular size variant of monoclonal antibodies (CE-SDS), which uses sodium dodecyl sulfate capillary electrophoresis (CE-SDS) with ultraviolet detection. Under reducing and non-reducing conditions, the purity of recombinant monoclonal antibody products is quantitatively determined according to molecular weight using capillary electrophoresis (2015 edition of the Chinese Pharmacopoeia 0542).

[0245] While the drug-to-antibody ratio has a precise value for a particular conjugate molecule (e.g., n in formula (I)), it should be understood that when used to describe samples containing many molecules, this value will often be an average, due to a degree of non-uniformity typically associated with the conjugation step. The average load of an immunoconjugate sample is referred herein to as the drug-to-antibody ratio or “DAR”. In some embodiments, the DAR is between about 1 and about 10, for example, 1–8, and typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. In some embodiments, at least 50% of the sample by weight is a compound having an average DAR plus or minus 2; in some specific embodiments, at least 50% of the sample is a conjugate containing an average DAR plus or minus 1. Examples include DAR values ​​of approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4. Immunoconjugates of approximately 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0. In some embodiments, 'approximately x' DAR means that the measured value of DAR is within 20% of x.

[0246] Methods for detecting DAR include, for example, extrapolating DAR values ​​from LC-MS data of reduced and deglycosylated samples. LC / MS allows quantification of the average number of drug-load molecules linked to the antibody in an ADC. HPLC separates antibodies into light and heavy chains, and further separates heavy chains (HC) and light chains (LC) based on the number of linker-load groups on each chain. Mass spectrometry data can identify the types of components in a mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. The average DAR of the ADC can be calculated based on the average loading of the LC and HC chains. The DAR of a given immunoconjugate sample represents the average number of drug (load) molecules linked to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.

[0247] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, such as an alkyl group containing 1 to 12 carbon atoms, an alkyl group containing 1 to 10 carbon atoms, or an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. For example, lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted; when substituted, the substituent can be substituted at any usable connection point. In non-limiting embodiments, the substituent is one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0248] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein the alkyl group is as defined above.

[0249] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, for example, an alkylene group containing 1 to 12 carbon atoms, or an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group can be substituted or unsubstituted, and when substituted, the substituent can be replaced at any usable connection point. In non-limiting embodiments, the substituent is independently selected from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0250] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0251] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 carbon atoms, for example, 3 to 12 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0252] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m(where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. For example, it may contain 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; or contain 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0253] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 members (e.g., 6 to 14 members, or 7 to 10 members) of monocyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups based on the number of shared spiroatoms between rings. For example, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic groups. Non-limiting examples of spirocyclic groups include:

[0254] The term "fused heterocyclic group" refers to a polycyclic heterocyclic group with 5 to 20 members (e.g., 6 to 14 members, or 7 to 10 members), in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Based on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, for example, 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0255] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group with 5 to 14 members (e.g., 6 to 14 members, or 7 to 10 members) that shares two non-directly connected atoms with any two rings. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m Bridged heterocyclic groups consist of heteroatoms (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. Based on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups. Non-limiting examples of bridged heterocyclic groups include:

[0256] The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0257] wait.

[0258] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0259] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, for example, a 6- to 10-membered group, such as phenyl or naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, and non-limiting examples include:

[0260] The aryl group can be substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0261] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. In non-limiting embodiments, the heteroaryl group is 5 to 10-membered, or 5- or 6-membered, such as furanyl, thiophene, pyridinyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0262] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0263] The term "amino protecting group" is used to protect the amino group by a group that is easily removed, so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, etc. These groups may optionally be substituted with 1 to 3 substituents selected from halogens, alkoxy groups, or nitro groups. For example, the amino protecting group is 9-fluorenylmethoxycarbonyl.

[0264] The term "cycloalkylalkyl" refers to an alkyl group that is substituted with one or more cycloalkyl groups, wherein the alkyl group is as defined above.

[0265] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0266] The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

[0267] The term "hydroxyl group" refers to the -OH group.

[0268] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0269] The term "amino" refers to -NH2.

[0270] The term "nitro" refers to -NO2.

[0271] The term "amide group" refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0272] The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0273] This disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterboranes, trideuterontetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0274] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0275] "Substituted" refers to one or more hydrogen atoms in a group, up to five in a non-limiting embodiment, for example, one to three hydrogen atoms independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0276] The term "anti-c-Met antibody" refers to an anti-c-Met antibody that is not conjugated to a heterologous module (such as a cytotoxic drug).

[0277] In this disclosure of anti-c-Met antibody-drug conjugates, "n" refers to the average amount of cytotoxic drug loaded onto each antibody or its antigen-binding fragment in the antibody-drug conjugate molecule. It can also be expressed as the ratio of drug amount to antibody amount, and is the average amount of drug in each ADC molecule after the conjugation reaction is identified by hydrophobic chromatography (HIC) mass spectrometry.

[0278] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0279] In this disclosure, the concentration of each substance in the pharmaceutical composition is calculated based on the total volume of the pharmaceutical composition. Detailed Implementation

[0280] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0281] Example 1. Design and preparation of antibody-drug conjugates

[0282] 1.1 Anti-c-Met antibody

[0283] The following anti-c-Met antibodies were prepared using standard antibody methods, such as vector construction, transfection into eukaryotic cells like HEK293 cells (Life Technologies Cat. No. 11625019), expression, separation, and purification.

[0284] The following is the sequence of the humanized anti-c-Met antibody:

[0285] Heavy chain:

[0286] Light chain:

[0287] 1.2. Preparation of Compounds 9A and 9B

[0288] Compounds 9A and 9B were prepared according to Example 9 in WO2020063676A:

[0289] N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-A

[0290] N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide 9-B

[0291] 1.3. Preparation of Antibody-Drug Conjugates (ADCs)

[0292] An antibody-drug conjugate (ADC) with the following structure was prepared, wherein Ab-10 is the anti-c-Met antibody prepared above.

[0293] At 37°C, add an appropriate amount of histidine-histidine hydrochloride buffer solution (10 mM histidine-histidine hydrochloride, pH = 5.8) to antibody Ab-10, followed by an appropriate amount of 200 mM EDTA (pH 6.3) to bring the antibody concentration to 15 g / L. Then add a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl) (10 mM, 14 eq), and react with shaking at 25°C for 22-24 hours, after which the reaction is stopped.

[0294] Compound 9-A (11 eq) was dissolved in dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25°C for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Capto S Impact cation exchange column (elution buffer: 50 mM HAc-NaAc buffer at pH 5.5 containing 390 mM NaCl) to obtain the exemplary product of the ADC formula (HAc-NaAc buffer), which was stored at 4°C.

[0295] The target DAR value was n = 6. The average drug loading calculated by RP-HPLC yielded a measured DAR value of n = 4.7-5.9.

[0296] Example 2. Screening of ADC buffer system (acetate)

[0297] Two buffer systems with different pH values ​​(10 mM acetate-sodium acetate, pH 5.0 and 5.5) were selected to prepare ADC formulation samples containing 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, and a protein concentration of 20.0 mg / mL. The thermal and colloidal stability of the antibody under different systems was investigated by measuring the melting temperature (Tm), thermal decomposition temperature (Tonset), aggregation temperature (Tagg), particle size (Radius), and dispersion factor (%PD). Simultaneously, the stability of the formulation under shaking, freeze-thaw, light exposure, room temperature (25℃), and high temperature (40℃) conditions was investigated by measuring the SEC-HPCL, reduced CE-SDS, and iCIEF purity of the samples.

[0298] 1) 10mM acetate-sodium acetate, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.0, 20 mg / mL ADC antibody

[0299] 2) 10mM acetate-sodium acetate, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.5, 20 mg / mL ADC antibody

[0300] Table 2 Screening Results of ADC Buffer System (Acetate) 1

[0301] Note: Multimodal indicates a diverse protein particle size distribution; the device cannot display the %PD value.

[0302] Table 3 Screening Results of ADC Buffer Systems (Acetate) 2

[0303] Table 4 Screening Results of ADC Buffer System (Acetate) 3

[0304] Note: In the table, NT indicates Not Tested.

[0305] Example 3. Screening of ADC buffer system (succinate)

[0306] Two buffer systems with different pH values ​​(10 mM succinic acid-sodium succinate, pH 5.0, 5.5, and 6.0) were used to prepare ADC formulation samples containing 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, and a protein concentration of 20.0 mg / mL. The thermal and colloidal stability of the antibody under different systems was investigated by measuring the melting temperature (Tm), thermal decomposition temperature (Tonset), aggregation temperature (Tagg), particle size (Radius), and dispersion factor (%PD). Simultaneously, the stability of the formulation under shaking, freeze-thaw, light exposure, room temperature (25°C), and high temperature (40°C) conditions was investigated by measuring the SEC-HPCL, reduced CE-SDS, and iCIEF purity of the samples.

[0307] 1) 10mM succinic acid-sodium succinate, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.0, 20 mg / mL ADC antibody

[0308] 2) 10mM succinic acid-sodium succinate, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.5, 20 mg / mL ADC antibody

[0309] 3) 10mM succinic acid-sodium succinate, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.0, 20 mg / mL ADC antibody

[0310] Table 5 Screening Results of ADC Buffer Systems (Succinate) 1

[0311] Table 6 Screening Results of ADC Buffer Systems (Succinate) 2

[0312] Table 7 Screening Results of ADC Buffer Systems (Succinate) 3

[0313] Example 4. Screening of ADC buffer system (histidine)

[0314] Three buffer systems with different pH values ​​(10 mM histidine-histidine hydrochloride, pH 5.5, 6.0, and 6.5) were used to prepare ADC formulation samples containing 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, and a protein concentration of 20.0 mg / mL. The thermal and colloidal stability of the antibody under different systems was investigated by measuring the melting temperature (Tm), thermal decomposition temperature (Tonset), aggregation temperature (Tagg), particle size (Radius), and dispersion factor (%PD). Simultaneously, the stability of the formulation under shaking, freeze-thaw, light exposure, room temperature (25℃), and high temperature (40℃) conditions was investigated by measuring the SEC-HPCL, reduced CE-SDS, and iCIEF purity of the samples.

[0315] 1) 10mM histidine-histidine hydrochloride, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.5, 20 mg / mL ADC antibody

[0316] 2) 10mM histidine-histidine hydrochloride, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.0, 20 mg / mL ADC antibody

[0317] 3) 10mM histidine-histidine hydrochloride, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.5, 20 mg / mL ADC antibody

[0318] Table 8 Screening Results of ADC Buffer System (Histidine) 1

[0319] Table 9. Screening results of ADC buffer system (histidine) 2

[0320] Table 10 Screening results of ADC buffer system (histidine) 3

[0321] The results of Examples 2-4 show:

[0322] The ADC antibody showed good appearance in 10 mM acetate-sodium acetate and 10 mM succinate-sodium succinate buffer systems. In a pH 5.0 10 mM acetate-sodium acetate buffer system, after 5 days under light and 4 weeks under high temperature, the ADC antibody showed a small increase in the SEC polymer ratio, a high sum of reduced CE-SDS light and heavy chain content, and a high percentage of iCIEF main peak area.

[0323] Example 5. Screening of ADC excipient types and pH ranges

[0324] An ADC formulation sample containing 20 mg / mL protein and different excipients (8% sucrose and 8% trehalose) was selected and freeze-dried using a 10 mM acetate-sodium acetate buffer system at pH 5.0 ± 0.3. The stability of the ADC stock solution under freeze-thaw, light, and 25℃, 40℃, and -35℃ conditions, as well as the stability of the finished formulation under light and 50℃ conditions, was investigated by measuring SEC-HPCL, reducing CE-SDS, iCIEF, DAR, free toxins, and binding activity.

[0325] 1) 10mM acetate-sodium acetate, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 4.7, 20 mg / mL ADC antibody

[0326] 2) 10mM acetate-sodium acetate, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.0, 20 mg / mL ADC antibody

[0327] 3) 10mM acetate-sodium acetate, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.3, 20 mg / mL ADC antibody

[0328] 4) 10mM acetate-sodium acetate, 8% (w / v) trehalose, 0.02% (w / v) polysorbate 80, pH 5.0, 20 mg / mL ADC antibody

[0329] Table 12 Screening Results of ADC Excipient Types and pH Ranges

[0330] Table 13 Screening Results of ADC Excipient Types and pH Ranges 2

[0331] Table 14 Screening Results of ADC Excipient Types and pH Ranges 3

[0332] The results of Example 5 show that:

[0333] There were no significant differences in any of the mass properties of the ADC samples at pH 0 under different pH conditions (4.7, 5.0, and 5.3). After being exposed to light and at a high temperature of 40°C, the increase in the proportion of SEC polymers in the stock solution and lyophilized powder under the low pH system (pH 4.7) was small, while the sum of the light and heavy chain contents of reduced CE-SDS and the percentage of the iCIEF main peak area were high. After being placed under freeze-thaw conditions and at -35°C, there was no significant difference in the purity of SEC monomers in the stock solution under the low pH system (pH 4.7) and pH 5.0 conditions compared to the zero point.

[0334] There were no significant differences in any of the mass properties of the ADC samples under different excipient systems (8% sucrose and 8% trehalose) at point 0. Under light and high temperature (40℃) conditions, the increase in the proportion of SEC polymers in the stock solution and lyophilized powder of the "10mM acetate-sodium acetate pH 4.7 + 8% sucrose + 0.02% polysorbate 80" system was small, while the sum of the light and heavy chain contents of reduced CE-SDS and the percentage of the iCIEF main peak area were high. Under freeze-thaw and -35℃ conditions, there were no significant differences in the purity of the stock solution SEC monomers in each group compared to point 0. At 50℃, there were no significant differences in any of the mass properties of the lyophilized powder (SEC-HPLC, reduced CE-SDS, iCIEF, DAR, free toxins, and binding activity) in each group compared to point 0.

[0335] Example 6. Preparation of freeze-dried powder

[0336] Different batches of ADC formulation samples were selected, containing 10 mM acetate-sodium acetate + 8% (w / v) sucrose + 0.02% polysorbate 80, pH 4.7, and a protein concentration of 20 g / L. These samples were pre-frozen and dried to obtain lyophilized powder. The stability of the finished formulation at 40°C was investigated by measuring the reconstitution time, moisture content, SEC-HPCL, iCIEF, DAR, and free toxins.

[0337] Table 15 Test Results of Freeze-Dried Powder

[0338] Table 16 Test Results of Freeze-Dried Powder 2

[0339] The results of Example 6 show that:

[0340] Compared with the sample at 0°C, there was no significant difference in the changes in SEC-HPLC purity, iCIEF purity, DAR and free toxin content of the lyophilized powder prepared using the formulation at 40°C.

[0341] Example 7. ADC Formulation

[0342] This invention also provides c-Met ADC antibody drug formulations with the following formulations, based on the total volume of the drug composition, including but not limited to:

[0343] (1) 10 mg / mL anti-c-Met antibody-drug conjugate, 10 mM acetate-sodium acetate, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 4.7;

[0344] (2) 20 mg / mL anti-c-Met antibody-drug conjugate, 10 mM acetate-sodium acetate, 8% (w / v) trehalose, 0.02% (w / v) polysorbate 80, pH 5.0;

[0345] (3) 20 mg / mL anti-c-Met antibody-drug conjugate, 20 mM acetate-sodium acetate, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 4.8;

[0346] (4) 30 mg / mL anti-c-Met antibody-drug conjugate, 5 mM acetate-sodium acetate, 8% (w / v) trehalose, 0.02% (w / v) polysorbate 80, pH 6.0;

[0347] (5) 20 mg / mL anti-c-Met antibody-drug conjugate, 10 mM acetate-sodium acetate, 2% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 4.0;

[0348] (6) 20 mg / mL anti-c-Met antibody-drug conjugate, 10 mM acetate-sodium acetate, 10% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 4.5;

[0349] (7) 40 mg / mL anti-c-Met antibody-drug conjugate, 10 mM acetate-sodium acetate, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 80, pH 4.2;

[0350] (8) 50 mg / mL anti-c-Met antibody-drug conjugate, 10 mM acetate-sodium acetate, 8% (w / v) trehalose, 0.05% (w / v) polysorbate 80, pH 5.5;

[0351] (9) 20 mg / mL anti-c-Met antibody-drug conjugate, 8% sucrose, 0.02% polysorbate 80, 10 mM acetic acid-sodium acetate, pH 4.7;

[0352] (10) 20 mg / mL anti-c-Met antibody-drug conjugate, 8% sucrose, 0.02% polysorbate 80, 10 mM acetic acid-sodium acetate, pH 5.0;

[0353] (11) 20 mg / mL anti-c-Met antibody-drug conjugate, 8% sucrose, 0.02% polysorbate 80, 10 mM acetic acid-sodium acetate, pH 5.2;

[0354] (12) 20 mg / mL anti-c-Met antibody-drug conjugate, 8% sucrose, 0.02% polysorbate 80, 10 mM acetate-sodium acetate, pH 4.2;

[0355] (13) 20 mg / mL anti-c-Met antibody-drug conjugate, 8% sucrose, 0.02% polysorbate 80, 10 mM acetate-sodium acetate, pH 5.7;

[0356] (14) 20 mg / mL anti-c-Met antibody-drug conjugate, 8% sucrose, 0.02% polysorbate 80, 10 mM acetic acid-sodium acetate, pH 5.3;

[0357] (15) 20 mg / mL anti-c-Met antibody-drug conjugate, 10 mM succinic acid-sodium succinate, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 4.7;

[0358] (16) 20 mg / mL anti-c-Met antibody-drug conjugate, 10 mM succinic acid-sodium succinate, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 5.0;

[0359] (17) 20 mg / mL anti-c-Met antibody-drug conjugate, 10 mM histidine-histidine hydrochloride, 8% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 6.0;

[0360] (18) 10 mg / mL anti-c-Met antibody-drug conjugate, 10 mM acetate-sodium acetate, 6% (w / v) sucrose, 0.02% (w / v) polysorbate 80, pH 4.7.

[0361] While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

Claims

1. A pharmaceutical composition comprising an anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof, and a buffer; The anti-c-Met antibody-drug conjugate has the structure shown in formula (I): Ab-(LYD)n Formula (I) in: Ab is an anti-c-Met antibody, which contains a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH contains HCDR1, HCDR2 and HCDR3 in any of the amino acid sequences shown in SEQ ID NO: 10-12, and the VL contains LCDR1, LCDR2 and LCDR3 in any of the amino acid sequences shown in SEQ ID NO: 13-15. The CDR is defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; - L- is a linker unit which is -L 1 - L 2 - L 3 - L 4 - L 1 It is -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, where W is selected from C. 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight-chain heteroalkyl with 1 to 8 atoms, wherein the heteroalkyl comprises 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 Integers from 1 to 20; L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - or chemical bond, where t is an integer from 1 to 6; R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl; R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups; Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b They may be the same or different, and each is independently selected from hydrogen, deuterium, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, or heterocyclic groups; or, R a and R b Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups; R 1 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups; Or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups; Or, R a and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups; m is an integer from 0 to 4; n is between 1 and 10, and n can be an integer or a decimal; D is a camptothecin-type drug, preferably eczema or its derivatives; The buffer is selected from one or more of acetate buffer, succinate buffer and histidine buffer; more preferably, it is selected from one or more of acetate-sodium acetate, succinate-sodium succinate and histidine-histidine hydrochloride.

2. The pharmaceutical composition according to claim 1, wherein the VH of the anti-c-Met antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1-3, and the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, 5 and 6, respectively; Preferably, 1) the VH of the anti-c-Met antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, 2, and 3, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 7, 5, and 6, respectively. Or 2) the anti-c-Met antibody VH contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, 2 and 3 respectively, and VL contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, 5 and 6 respectively; The CDR is defined according to the Kabat system; Preferably, the anti-c-Met antibody comprises any one of the following: (1) The VH contains an amino acid sequence shown in SEQ ID NO: 11 or having at least 90% sequence identity with it, and the VL contains an amino acid sequence shown in SEQ ID NO: 14 or having at least 90% sequence identity with it; (2) The VH contains an amino acid sequence shown in SEQ ID NO: 10 or having at least 90% sequence identity with it, and the VL contains an amino acid sequence shown in SEQ ID NO: 13 or having at least 90% sequence identity with it; (3) The VH contains an amino acid sequence shown in SEQ ID NO: 12 or having at least 90% sequence identity with it, and the VL contains an amino acid sequence shown in SEQ ID NO: 15 or having at least 90% sequence identity with it.

3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-c-Met antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, preferably a humanized antibody; Preferably, the anti-c-Met antibody further comprises an Fc region; Preferably, the Fc region is derived from the Fc region of human IgG1, IgG2, IgG3 or IgG4; more preferably, it is derived from the Fc region of human IgG2.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the anti-c-Met antibody comprises a combination selected from the following light chains and heavy chains: The heavy chain comprises an amino acid sequence shown in SEQ ID NO: 18 or having at least 90% sequence identity with it, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 19 or having at least 90% sequence identity with it; The heavy chain comprises an amino acid sequence shown in SEQ ID NO: 16 or having at least 90% sequence identity with it, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 17 or having at least 90% sequence identity with it; or, The heavy chain comprises an amino acid sequence shown in or having at least 90% sequence identity with SEQ ID NO: 20, and the light chain comprises an amino acid sequence shown in or having at least 90% sequence identity with SEQ ID NO:

21.

5. The pharmaceutical composition according to any one of claims 1-4, wherein n is 1 to 8, and n is a decimal or an integer. Preferably, n is an integer or decimal from 3 to 7. More preferably, n is an integer or decimal of 4 to 6.

6. The pharmaceutical composition according to any one of claims 1-5, wherein it is an antibody-drug conjugate of formula II or a pharmaceutically acceptable salt thereof: in: W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight-chain heteroalkyl with 1 to 8 atoms, wherein the heteroalkyl comprises 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 Integers from 1 to 20; L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl; R 1 Selected from hydrogen atoms, halogens, cycloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups; Or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups; R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl; R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups; m is an integer from 0 to 4; n is between 1 and 10, and n can be an integer or a decimal; Ab is defined as in any one of claims 1-5.

7. The pharmaceutical composition according to any one of claims 1-6, wherein: The -LY- structure is as follows: s 1 Integers between 2 and 8; L 2 L 3 R 1 R 2 R 5 R 6 R 7 and m as defined in claim 6.

8. The pharmaceutical composition according to any one of claims 1-7, wherein it is an antibody-drug conjugate of general formula (III) or a pharmaceutically acceptable salt or solvate thereof: in: s 1 Integers from 2 to 8; preferably 5; Ab、R 1 R 2 R 5 R 6 R 7 m and n are as defined in claim 6.

9. The pharmaceutical composition according to any one of claims 1-8, wherein -L- is: Or, among them -LY- is selected from: Preferred 10. The pharmaceutical composition according to any one of claims 1-9, wherein the anti-c-Met antibody-drug conjugate has the structure shown in the following formula: in: n is an integer or decimal from 1 to 10, and Ab-10 is an anti-c-Met antibody; Preferably, 1) the VH of the anti-c-Met antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, 2 and 3 respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 7, 5 and 6 respectively, wherein the CDR is defined according to the Kabat system; Preferably, the anti-c-Met antibody VH comprises the amino acid sequence shown in SEQ ID NO: 11 or having at least 90% sequence identity with it, and the VL comprises the amino acid sequence shown in SEQ ID NO: 14 or having at least 90% sequence identity with it; More preferably, the anti-c-Met antibody heavy chain comprises an amino acid sequence shown in SEQ ID NO: 18 or having at least 90% sequence identity with it, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 19 or having at least 90% sequence identity with it.

11. The pharmaceutical composition according to any one of claims 1-10, wherein the pH value of the buffer or the pharmaceutical composition is from about 3.0 to about 8.0, preferably from about 4.0 to about 7.0, more preferably from about 4.0 to about 6.5, and more preferably 4.7±0.5 or 5.2±0.

5.

12. The pharmaceutical composition according to any one of claims 1-11, wherein the concentration of the buffer is from about 1 mM to about 100 mM, preferably from about 5 mM to about 40 mM, more preferably from about 5 mM to about 20 mM.

13. The pharmaceutical composition according to any one of claims 1-12, wherein the concentration of the anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof is from about 0.01 mg / mL to about 200 mg / mL, preferably from about 0.1 mg / mL to about 160 mg / mL, more preferably from about 1 mg / mL to about 100 mg / mL.

14. The pharmaceutical composition according to any one of claims 1-13, further comprising a surfactant; Preferably, the surfactant is polysorbate, more preferably polysorbate 20 or polysorbate 80, and more preferably polysorbate 80; Preferably, the concentration of the surfactant is about 0.01 to 0.10% (w / v), more preferably about 0.01 to 0.05% (w / v).

15. The pharmaceutical composition according to any one of claims 1-14, further comprising carbohydrates; Preferably, the sugar is sucrose or trehalose; Preferably, the concentration of the sugar is from about 0.1% (w / v) to about 20% (w / v), more preferably from about 2% (w / v) to about 12% (w / v), and even more preferably from about 4% (w / v) to about 10% (w / v).

16. The pharmaceutical composition according to any one of claims 1-15, comprising: (A) Approximately 1–100 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 5–20 mM acetic acid-sodium acetate, Approximately 2–10% (w / v) sucrose or trehalose, Approximately 0.01–0.05% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 4.0–6.0; or (B) Approximately 1–100 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 5–20 mM succinic acid-sodium succinate, Approximately 2-10% (w / v) sucrose, Approximately 0.01–0.05% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 5.0–6.0; or (C) Approximately 1–100 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 5–20 mM histidine-histidine hydrochloride. Approximately 2-10% (w / v) sucrose, Approximately 0.01–0.05% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 5.5–6.5; Preferably, the pharmaceutical composition comprises: (A1) Approximately 20 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 10 mM acetic acid-sodium acetate, Approximately 6% (w / v) sucrose, Approximately 0.02% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 4.6–5.5; (A2) Approximately 20 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 10 mM acetic acid-sodium acetate, Approximately 8% (w / v) sucrose, Approximately 0.02% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 4.2 to 5.8, preferably 4.7±0.5, 5.0±0.5, 5.2±0.5 or 5.3±0.5; (A3) Approximately 20 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 10 mM acetic acid-sodium acetate, Approximately 8% (w / v) trehalose, Approximately 0.02% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 4.6 to 5.3, preferably 5.0; (B1) Approximately 20 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 10 mM succinic acid-sodium succinate, Approximately 6% (w / v) sucrose, Approximately 0.02% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 5.0 to 6.0, preferably 5.0, 5.5 or 6.0; or, (C1) Approximately 20 mg / mL of anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof. Approximately 10 mM histidine-histidine hydrochloride. Approximately 6% (w / v) sucrose, Approximately 0.02% (w / v) polysorbate 80, The pH of the pharmaceutical composition is 5.5 to 6.5, preferably 5.5, 6.0 or 6.

5.

17. A lyophilized formulation obtained by freeze-drying a liquid formulation comprising the pharmaceutical composition of any one of claims 1-16.

18. A reconstituted solution obtained by reconstituted the lyophilized formulation of claim 17.

19. An article comprising a container containing a pharmaceutical composition as claimed in any one of claims 1 to 16, a lyophilized formulation as claimed in claim 17, or a reconstituted solution as claimed in claim 18.

20. Use of the pharmaceutical composition of any one of claims 1 to 16, the lyophilized formulation of claim 17, the reconstituted solution of claim 18, or the article of claim 19 in the preparation of a medicament for the prevention or treatment of a disease; Preferably, the disease or condition is cancer, or a c-Met-mediated disease or condition; More preferably, the cancer is a cancer expressing c-Met; Ideally, the cancer is stomach cancer, lung cancer, intestinal cancer, or liver cancer.

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