Antibody-drug conjugates, methods for preparing them, and their use

Abstract

This invention provides an antibody-drug conjugate, a method for preparing the same, and its use, and belongs to the pharmaceutical field. The structure of this antibody-drug conjugate is as shown in Formula I. In the formula, antibody Ab is an anti-HER2 antibody, q is an integer from 1 to 20, and D is the drug portion. The antibody-drug conjugate provided in this invention has a low aggregate content and a low proportion of naked antibody, and an appropriate DAR value, so it exhibits excellent plasma stability and antitumor effect, can effectively suppress tumor regrowth, and has great potential for clinical application. TIFF2026513895000034.tif51170

Description

[Technical Field]

[0001] This invention belongs to the pharmaceutical field and, more specifically, relates to an antibody-drug conjugate using an anti-HER2 antibody as a targeting linker, a method for preparing the same, and its use. [Background technology]

[0002] Antibody-drug conjugates (ADCs) have ushered in a new era of cancer treatment by selectively delivering drugs to cancer cells and killing them while having minimal impact on normal cells. Several ADC drugs have been approved by the FDA and are currently marketed, including Mylotarg, which combines the CD33 antibody with calichemycin; Adcetris, which combines the CD30 antibody with auristatin E and is used to treat Hodgkin lymphoma and anaplastic large cell lymphoma patients; DS-8201, which combines the HER2 antibody with the camptothecin derivative Dxd and is used to treat HER2-positive breast cancer patients; and sacituzumab govitecan, which targets the TROP-2 antigen (also known as epithelial glycoprotein 1, EGP-1) and is used in triple-negative breast cancer.

[0003] To date, the drugs contained in ADCs approved by the FDA primarily target DNA or tubulin. Camptothecin derivatives (SN-38, Dxd, Dx-8951, etc.), known to exert their antitumor effects by inhibiting DNA topoisomerase I, have been shown to exhibit potent antitumor effects against multiple cancer cell types both in vitro and in vivo. Compounds that inhibit tubulin, such as eribulin, MMAE, MMAF, and maytansine, similarly exhibit potent antitumor effects against multiple cancer cell types both in vitro and in vivo. The structural formulas of compounds known to act on DNA or tubulin are as follows: JPEG2026513895000002.jpg101170.

[0004] Based on the mechanism of action of conventional ADC drugs, antibody-drug conjugates specifically bind to cell surface proteins, and the resulting conjugates are taken up into cells, thereby delivering drug molecules to the target cells. Therefore, the intracellular drug concentration is directly related to the distribution density of targets on the cell surface that can be specifically recognized by the antibody. However, the density of targets that can be recognized by the antibody on the molecular surface is usually low, which leads to the problem of low drug concentrations in target cells. To solve this problem, a currently widely used method is to increase the drug-antibody ratio (DAR) of the ADC, thereby increasing the amount of drug that enters the cell. However, according to a study by Hamblett et al. (Clin Cancer Res. 2004, 10, 7063), increasing the DAR value of ADCs produced unexpected effects in pharmacokinetic exposure, such as severe aggregation of ADC molecules, decreased stability, and an increase in low-molecular-weight toxins in the blood, resulting in toxicity and side effects.

[0005] Since 2013, Daiichi Sankyo Co., Ltd. has filed numerous patent applications (CN201380053256.2, CN201910768778.X, CN201980061665.4, etc.) disclosing a series of antibody-drug conjugates having specific linker-low molecular weight toxin structures, and specifically disclosing ADCs with the following typical structures that exhibit excellent efficacy. However, further improvements in their stability and therapeutic efficacy are expected. Therefore, the development of drug conjugates that can target lesion sites, significantly improve aggregation phenomena, enhance stability and therapeutic efficacy, is extremely important. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide an antibody-drug conjugate using an anti-HER2 antibody as a targeting linker, a method for preparing the same, and its use. [Means for solving the problem]

[0007] The present invention provides an antibody-drug conjugate, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof, wherein the antibody-drug conjugate has the structure shown by formula I: In formula TIFF2026513895000004.tif44170, Ab is an anti-HER2 antibody, and the anti-HER2 antibody is either Inetetamab or trastuzumab. q is an integer between 1 and 20. D is the drug portion.

[0008] Furthermore, D is a cytotoxic agent, a treatment for autoimmune toxicity, or an anti-inflammatory agent. Preferably, D is a drug that targets DNA or a drug that targets tubulin. More preferably, D is It is one of the compounds or derivatives thereof from JPEG2026513895000005.jpg104170.

[0009] Furthermore, the structure of the antibody-drug conjugate is as shown in Formula II: In formula TIFF2026513895000006.tif65170, Ab is an anti-HER2 antibody, and the anti-HER2 antibody is either Inetetamab or trastuzumab. q is an integer between 1 and 20.

[0010] Furthermore, the heavy chain sequence of Inetetamab is as shown in SEQ ID NO:1, and the light chain sequence is as shown in SEQ ID NO:2.

[0011] Furthermore, the heavy chain sequence of trastuzumab is as shown in SEQ ID NO:3, and the light chain sequence is as shown in SEQ ID NO:4.

[0012] Furthermore, the DAR value of the antibody-drug conjugate is from 1.00 to 20.00, preferably from 2.0 to 8.0.

[0013] Furthermore, the DAR value of the antibody-drug conjugate is from 2.0 to 5.0, preferably 4.08 ± 0.5.

[0014] The present invention further provides a pharmaceutical preparation for preventing and / or treating tumors, wherein the pharmaceutical preparation is prepared by adding a pharmaceutically acceptable excipient, with the aforementioned antibody-drug conjugate, or its stereoisomer, or its optical isomer, or its salt, or its deuterated form as an active ingredient.

[0015] Furthermore, the preparation is an oral preparation or an injection preparation.

[0016] The present invention also provides the use of the aforementioned antibody-drug conjugate, or its stereoisomer, or its optical isomer, or its salt, or its deuterated form in the preparation of a pharmaceutical preparation for preventing and / or treating tumors.

[0017] Furthermore, the tumor is selected from lung cancer, urinary tract cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma, cholangiocarcinoma.

[0018] The present invention further provides a method for preparing the aforementioned antibody-drug conjugate, or its stereoisomer, or its optical isomer, or its salt, or its deuterated form, the method including the step of obtaining the antibody-drug conjugate by coupling an anti-HER2 antibody with Compound III, and the structure of the Compound III is as follows: TIFF2026513895000007.tif36170D is the aforementioned drug moiety.

[0019] Regarding the definitions of terms in the present invention, unless otherwise specified, the initial definitions given to radicals or terms in this specification apply throughout this specification to said radicals or terms, and for terms not specifically defined in this specification, they shall have the meanings that can be imparted by those skilled in the art based on the disclosure content and context.

[0020] The targeted linker-drug conjugate refers to a conjugate obtained by conjugating a drug with a targeted linker having a targeting binding effect to a lesion site through a linker. For example, an antibody-drug conjugate (ADC) is a type of targeted linker-drug conjugate, and its targeted linker is an antibody or an antibody fragment.

[0021] The drug-linking building unit is a product obtained by linking the linker and the drug in the aforementioned targeted linker-drug conjugate, and is an intermediate for preparing the targeted linker-drug conjugate. By conjugating it with an antibody, an antibody-drug conjugate can be obtained.

[0022] In the present invention, the DAR value represents the average number of drug-linking building units conjugated to one antibody in an antibody-drug conjugate, and corresponds to the average value of the q value. The DAR value may not be an integer.

[0023] The deuterated compound refers to a compound in which one or more hydrogen atoms in the compound are replaced by deuterium.

[0024] A linker (also known as a binding agent) is a substance used to link a compound having a therapeutic effect with a targeted linker having a targeting binding effect to a lesion site. A linker fragment refers to a partial structure having a linking function present in the linker. <00001​​​​​ In the case of a targeted linker-drug conjugate containing a non-cleavable linker (e.g., an antibody-drug conjugate), the drug release mechanism may involve the conjugate binding to the antigen and being taken up into the cell, after which the antibody is enzymatically degraded in lysosomes, releasing an active small molecule compound composed of the drug, the linker, and antibody amino acid residues.

[0027] Furthermore, targeted linker-drug conjugates containing cleavable linkers (e.g., antibody-drug conjugates) can cleave within target cells and release the drug (e.g., the drug itself in the case of small molecule drugs). Cleavable linkers are classified into two main categories: chemically unstable linkers and enzymatically unstable linkers. Chemically unstable linkers selectively cleave depending on the properties of the plasma and cytoplasm (pH value, glutathione concentration, etc.). Enzymatically unstable linkers (e.g., peptide linkers) can be efficiently cleaved by lysosomal proteases such as cathepsin and plasmin. Peptide linkers are considered to be extremely stable in plasma.

[0028] Linkers constitute the core component of targeted linker-drug conjugates (e.g., antibody-drug conjugates) and can significantly influence the pharmacokinetics, therapeutic index, and efficacy of such conjugates.

[0029] This invention relates to the linker in By creatively introducing the structure shown in TIFF2026513895000008.tif21170, it is possible to significantly improve the safety, stability, efficacy, and controllability of the resulting antibody-drug conjugate while retaining the conventional linker function, thereby effectively promoting the clinical application of antibody-drug conjugate pharmaceuticals.

[0030] The antibody-drug conjugate provided in the present invention comprises an anti-HER2 antibody and 1 to 20 covalently bound drug-linking construct units, the drug-linking construct units may be linked via binding to thiol groups derived from interchain disulfide bonds in the reducing antibody and / or binding to thiol groups derived from cysteine ​​residues.

[0031] To facilitate linking, drug linkage constructs are usually constructed before linking with the anti-HER2 antibody. However, the construction order can be changed. For example, a method can be employed in which a construct with a protecting group is first linked to the anti-HER2 antibody, and then the protecting group is removed and other drug units are added after linking to the anti-HER2 antibody.

[0032] The antibody-drug conjugate provided in the present invention targets tumor cells expressing HER2, and after binding to HER2 on the cell surface, the conjugate enters the cell by intracellular uptake, and the drug is released into the cell in an active form to exert its pharmacological effect, or the drug is released extracellularly, and the drug penetrates into the cell to exert its therapeutic effect. [Effects of the Invention]

[0033] The beneficial effects of this invention are as follows:

[0034] This invention relates to linkers. By introducing the structure shown in TIFF2026513895000009.tif21170, it is possible to reduce the aggregate content and naked antibody percentage of the antibody-drug conjugate, which is composed of a linker, drug unit, and anti-HER2 antibody linked together, maintain the DAR value of the antibody-drug conjugate within an appropriate range, enhance the plasma stability of the antibody-drug conjugate, and improve the therapeutic effect. The final prepared antibody-drug conjugate exhibits excellent plasma stability and antitumor efficacy due to its low aggregate content, low proportion of unadulterated antibodies, and appropriate DAR value. It can effectively suppress tumor regrowth and is highly promising for clinical application.

[0035] Furthermore, it will be obvious to those skilled in the art that, based on the above-described content of the present invention, various other forms of modification, substitution, or alteration can be made using ordinary technical knowledge and conventional means in the aforementioned technical field, without departing from the above-described basic technical concept of the present invention. The above-mentioned aspects of the present invention will be further described below with reference to specific embodiments of the examples. However, these should not be understood as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above-mentioned aspects of the present invention fall within the scope of the present invention. [Brief explanation of the drawing]

[0036] [Figure 1] A diagram showing the stability of ADC in mouse, dog, monkey, and human plasma. [Figure 2] Figure showing the antitumor effects of ADC in tumor models of ovarian cancer (SKOV-3), gastric cancer (NCI-N87), pancreatic cancer (Capan-1), breast cancer (JIMT-1), and breast cancer (HCC1954). [Figure 3] A diagram showing the sustained in vivo tumor-suppressing effect of ADCs. [Modes for carrying out the invention]

[0037] All raw materials and equipment used in this invention are known products and were obtained by purchasing commercially available products.

[0038] Example 1: Preparation of antibody-drug conjugate (SMP-656) I. Preparation of Intermediate A TIFF2026513895000010.tif65170 Step 1: Preparation of Compound A-2 TIFF2026513895000011.tif29170A-1 (20.0 g, 56.49 mmol) and acetonitrile (200 mL) were placed in a 500 mL flask and stirred at room temperature. After complete clarity, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (11.0 g, 57.38 mmol) and N-hydroxysuccinimide (7.0 g, 60.82 mmol) were added, and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by TLC. The reaction mixture was filtered, and the resulting solid was vacuum-dried to obtain 22.0 g of compound A-2 (86% yield).

[0039] Step 2: Preparation of Compound A-3 TIFF2026513895000012.tif38170L-phenylalanine (8.0 g, 48.48 mmol), sodium bicarbonate (8.0 g, 95.24 mmol), and water (200 mL) were placed in a 500 mL flask and stirred at room temperature. After complete clarity, compound A-2 (22.0 g, 48.78 mmol) was dissolved in diglyme (50 mL) and slowly added dropwise to the reaction mixture. After addition, the reaction was continued with stirring at room temperature for 12 hours, and completion of the reaction was confirmed by TLC. Diglyme was removed by distillation under reduced pressure, and the residual reaction solution was added dropwise to 0.5 M hydrochloric acid aqueous solution (500 mL), resulting in the precipitation of a large amount of solid. After filtration, the obtained solid was vacuum-dried to obtain 15.0 g of compound A-3 (yield 61%). 1 HNMR (400 MHz, CDCl3) 12.51 (s, 1H), 9.04 (s, 1H), 8.31 (s, 1H), 7.95 (s, 1H), 7.90(d, J=8.0 Hz, 2H), 7.56(d, J=7.8 Hz, 2H), 7.38-7.28 (m, 4H), 7.19-7.14(m, 5H), 4.85(t, J=8.2 Hz, 1H), 4.71(d, J=8.2 Hz, 2H), 4.39(t, J=8.4 Hz, 1H), 4.10-3.83(m, 4H), 3.12(d, J=9.6 Hz, 1H), 2.85(d, J=9.6 Hz, 1H).

[0040] Step 3: Preparation of compound A-4 In a 500 mL flask, A-3 (15.0 g, 29.94 mmol), acetonitrile (200 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (6 g, 31.30 mmol), and N-hydroxysuccinimide (4 g, 34.78 mmol) were sequentially added, and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by TLC. The reaction mixture was filtered, and the resulting solid was vacuum-dried to obtain 13.0 g of compound A-4 (73% yield).

[0041] Step 4: Preparation of compound A-5 TIFF2026513895000014.tif31170 Glycine (3.0 g, 40.00 mmol), sodium bicarbonate (6.7 g, 79.76 mmol), and water (150 mL) were placed in a 500 mL flask and stirred at room temperature. After it became completely clear, compound A-4 (13.0 g, 21.74 mmol) was dissolved in diglyme (40 mL) and slowly added dropwise to the reaction mixture. After the addition was complete, the reaction was continued with stirring at room temperature for 12 hours, and the completion of the reaction was confirmed by TLC. The mixture was concentrated under reduced pressure, and the organic solvent was removed by distillation. The residual reaction mixture was added dropwise to 0.5 M hydrochloric acid aqueous solution (300 mL), and a large amount of solid precipitated. After filtration, the obtained solid was vacuum dried to obtain 10.0 g of compound A-5 (yield 83%). 1 HNMR (400 MHz, CDCl3) 13.01 (s, 1H), 9.01 (s, 1H), 8.27 (s, 1H), 7.98 (s, 1H), 7.89(d, J=8.0 Hz, 2H), 7.54(d, J=7.8 Hz, 2H), 7.34-7.23 (m, 4H), 7.19-7.14(m, 5H), 4.75(t, J=8.2 Hz, 1H), 4.61(d, J=8.2 Hz, 2H), 4.30(t, J=8.4 Hz, 1H), 4.04-3.83(m, 6H), 3.10(d, J=9.6 Hz, 1H), 2.75(d, J=9.6 Hz, 1H).

[0042] Step 5: Preparation of compound A-6 In a 25 mL flask, A-5 (200.0 mg, 0.36 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (163.0 mg, 0.43 mmol), N,N-diisopropylethylamine (66.9 mg, 0.52 mmol), and N,N-dimethylformamide (5 mL) were sequentially added. After stirring at room temperature for 5 minutes, eribulin (263.1 mg, 0.36 mmol) was added. The reaction was continued with stirring at room temperature for 10 minutes, and completion of the reaction was confirmed by TLC. N,N-dimethylformamide was removed by distillation under reduced pressure, and the residue was purified by thin-layer chromatography (dichloromethane / methanol = 10:1) to obtain 300.0 mg of compound A-6 (yield 66%).

[0043] Step 6: Preparation of Compound A TIFF2026513895000016.tif3917025 mL flask was sequentially mixed with N,N-dimethylformamide (2 mL), A-6 (20.0 mg, 0.016 mmol), and 1,8-diazabicyclo[5.4.0]undeca-7-ene (5.0 mg, 0.032 mmol). The mixture was stirred at room temperature for 30 minutes, and the completion of the reaction was confirmed by TLC. N,N-dimethylformamide was removed by distillation under reduced pressure, and the residue was purified by preparative HPLC to obtain 10.0 mg of compound A (60% yield). 1HNMR (400 MHz, DMSO-d6) δ 8.48 (t, J = 5.5 Hz, 1H), 8.31 (dd, J = 10.1, 4.6 Hz, 2H), 7.98 (s, 3H), 7.74 (t, J = 5.6 Hz, 1H), 7.30 ― 7.21 (m, 4H), 7.22 ― 7.15 (m, 1H), 5.02 (d, J = 22.6 Hz, 2H), 4.79 (d, J = 29.3 Hz, 2H), 4.63 (dd, J = 5.7, 3.7 Hz, 1H), 4.59 ― 4.50 (m, 2H), 4.24 (t, J = 13.5 Hz, 1H), 4.21 ― 4.12 (m, 1H), 4.12 ― 4.06 (m, 3H), 4.02 (s, 1H), 3.86 (dd, J = 16.8, 5.7 Hz, 1H), 3.68 (dd, J = 16.7, 5.5 Hz, 4H), 3.54 (dd, J = 19.0, 7.3 Hz, 12H), 3.11 (dd, J = 16.3, 11.0 Hz, 1H), 3.07 ― 2.99 (m, 2H), 2.84 (d, J = 9.6 Hz, 1H), 2.80 ― 2.65 (m, 3H), 2.62 ― 2.54 (m, 1H), 2.24 (ddd, J = 48.1, 23.9, 9.6 Hz, 6H), 1.98 (dd, J = 29.5, 15.1 Hz, 6H), 1.73 ― 1.57 (m, 5H), 1.55 ― 1.40 (m, 2H), 1.37 ― 1.25 (m, 3H), 1.22 ― 1.13 (m, 1H), 1.03 (d, J = 6.4 Hz, 3H), 1.00 ― 0.89 (m, 1H).

[0044] II. Modulation of compound SMP-93566 TIFF2026513895000017.tif54170 Project 1: Modulation of compound SMP-93566-2 In a 100 mL flask, intermediate SMP-93566-1 (1.0 g, 5.75 mmol), tert-butyl bromoacetate (0.9 g, 4.60 mmol), potassium carbonate (0.6 g, 4.60 mmol), and N,N-dimethylformamide (20 mL) were sequentially added and reacted at room temperature for 2 hours. After confirming that the main product had the molecular weight of the target compound by LC-MS analysis, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (dichloromethane / methanol = 12:1) to obtain 0.2 g of compound SMP-93566-2 (yield 15.1%). MS (ESI) m / z: 289[M+H] + . 1 HNMR (400 MHz, CDCl3) δ5.91 (s, 1H), 3.40-3.49(m, 8H), 323 (s, 2H), 2.54(m, 4H), 2.70(m, 4H),1.33 (s, 9H).

[0045] Step 2: Preparation of compound SMP-93566-3 TIFF2026513895000019.tif39170 In a 10 mL flask, intermediate SMP-93566-2 (200.0 mg, 0.69 mmol), maleimidopropionic acid (140.8 mg, 0.83 mmol), N,N-dimethylformamide (5 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate ( 315.4 mg (0.83 mmol) and N,N-diisopropylethylamine (134.2 mg, 1.04 mmol) were sequentially added, and the mixture was reacted at room temperature for 30 minutes. After confirming that the main product had the molecular weight of the target compound by LC-MS analysis, the reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC to obtain 220.0 mg (72.6% yield) of compound SMP-93566-3. MS (ESI) m / z: 440[M+H] + .

[0046] Step 3: Preparation of Compound SMP-93566-4 In a 3217025 mL flask, SMP-93566-3 (220.0 mg, 0.50 mmol), dichloromethane (5 mL), and trifluoroacetic acid (5 mL) were sequentially added and reacted at room temperature for 2 hours. After confirming by LC-MS analysis that the main product had the molecular weight of the target compound, the reaction solution was concentrated under reduced pressure to obtain a crude product. A small amount of acetonitrile was added to the crude product to dissolve it, and it was purified by preparative HPLC to obtain 90.0 mg (yield 47.0%) of compound SMP-93566-4. MS (ESI) m / z: 384[M+H] + .

[0047] Step 4: Preparation of Compound SMP-93566 In a 3817010 mL flask, SMP-93566-4 (8.8 mg, 0.023 mmol), intermediate A (20 mg, 0.019 mmol), N,N-dimethylformamide (2 mL), O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (8.7 mg, 0.023 mmol), and N,N-diisopropylethylamine (3.7 mg, 0.029 mmol) were sequentially added and reacted at room temperature for 20 minutes. After confirming by LC-MS analysis that the main product had the molecular weight of the target compound, the reaction solution was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by preparative HPLC to obtain 18.0 mg (yield 67.0%) of compound SMP-93566. MS (ESI) m / z: 1414[M+H] + ,707[M / 2+H] + . 1H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H), 8.29 (t, J = 5.6 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.12 (t, J = 5.6 Hz, 1H), 7.95 (s, 1H), 7.72 (s, 1H), 7.31 ― 7.09 (m, 6H), 6.98 (s, 2H), 5.02 (d, J = 21.4 Hz, 2H), 4.82 (s, 1H), 4.75 (s, 1H), 4.63 (s, 1H), 4.58 ― 4.48 (m, 2H), 4.28 ― 4.06 (m, 10H), 3.87 - 3.47 (m, 30H), 3.16 - 2.99 (m, 5H), 2.85 - 2.64 (m, 6H), 2.62 - 2.53 (m, 2H), 2.34 - 2.16 (m, 6H), 2.01 - 1.87 (m, 6H), 1.73 ― 1.58 (m, 6H), 1.31 (s, 3H), 1.19 (m, 1H), 1.03 (d, J = 6.3 Hz, 3H), 1.00 ― 0.91 (m, 1H).

[0048] III. Preparation of Antibody-Drug Conjugates (SMP-656) Coupling Method: Antibody-drug conjugates were prepared by drug coupling utilizing the disulfide bonds of the antibody. First, the antibody was prepared in a solution to a concentration of 20 mg / mL. 0.1 mL of the antibody solution was transferred to a 1.5 mL centrifuge tube, PBS (pH 7.4) / DTPA solution (293 μL) was added, followed by 5 mM TCEP aqueous solution (6.4 μL, 2.4 equivalents per antibody molecule). The mixture was incubated at 25°C for 2 hours to reduce the disulfide bonds in the antibody hinge region to sulfhydryl groups. Subsequently, 10% medical DMSO and 10 mM payload solution (13.3 μL, 10 equivalents per antibody molecule) were added to the aforementioned solution. The mixture was reacted at 25°C under shaking conditions of 400 rpm for 2 hours to link the drug-linking construct to the antibody, thereby obtaining the antibody-drug conjugate.

[0049] The antibody-drug conjugate obtained by applying the aforementioned coupling method to an anti-HER2 antibody using compound SMP-93566 as the drug-linking constructor was named SMP-656.

[0050] Concentration of antibody-drug conjugate: The obtained antibody-drug conjugate solution was transferred to a 5000K (Millipore) ultrafiltration tube and centrifuged at 2°C and 3500G for 10 minutes using a high-speed cryogenic centrifuge (GENESPEED 1580R).

[0051] The sequence of the anti-HER2 antibody used in this example is as follows: Inetetamab Heavy chain (SEQ ID NO:1): EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSSASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPG; Light chain (SEQ ID NO:2): DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPSRFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC.

[0052] The method for preparing the control ADC sample is shown below.

[0053] Comparative Example 1: Preparation of ADC (DS-8201a) corresponding to DS-8201 I. Preparation of compound DS-8201 TIFF2026513895000022.tif62170 Step 1: Preparation of compound DS-8201 In a 10 mL flask, intermediate B (50.0 mg, 0.06 mmol), maleimidohexanoic acid (12.6 mg, 0.06 mmol), and N,N-dimethylformamide (2 mL) were added sequentially. After complete dissolution, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.2 mg, 0.06 mmol) and N,N-diisopropylethylamine (23.2 mg, 0.18 mmol) were added sequentially. The reaction was stirred at room temperature for 20 minutes, and after confirming completion of the reaction by TLC, the reaction mixture was purified by preparative HPLC to obtain 35 mg of compound DS-8201 (yield 56.4%). MS (ESI) m / z: 1034[M+H] + .

[0054] II. Preparation of DS-8201a Coupling Method: Antibody-drug conjugates were prepared by drug coupling utilizing the disulfide bonds of the antibody. First, the antibody was prepared in a solution to a concentration of 20 mg / mL. 0.1 mL of the antibody solution was transferred to a 1.5 mL centrifuge tube, PBS (pH 7.4) / DTPA solution (293 μL) was added, followed by 5 mM TCEP aqueous solution (16.0 μL, 6.0 equivalents per antibody molecule). The mixture was incubated at 25°C for 2 hours to reduce the disulfide bonds in the antibody hinge region to sulfhydryl groups. Subsequently, 10% medical DMSO and 10 mM payload solution (13.3 μL, 10 equivalents per antibody molecule) were added to the aforementioned solution. The mixture was reacted at 25°C under shaking conditions of 400 rpm for 2 hours to link the drug-linking construct to the antibody, thereby obtaining the antibody-drug conjugate.

[0055] The antibody-drug conjugate obtained by applying the aforementioned coupling method to an anti-HER2 antibody using compound DS-8201 as a drug-linking constructor was named DS-8201a.

[0056] Concentration of antibody-drug conjugate: The obtained antibody-drug conjugate solution was transferred to a 5000K (Millipore) ultrafiltration tube and centrifuged at 2°C and 3500G for 10 minutes using a high-speed cryogenic centrifuge (GENESPEED 1580R).

[0057] The sequence of the anti-HER2 antibody used in this comparative example is as follows: Trastuzumab Heavy chain (SEQ ID NO:3): EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRYADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSSASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPG; Light chain (SEQ ID NO:4): DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPSRFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC.

[0058] Comparative Example 2: Preparation of an ADC (SMP-79786-X) corresponding to SMP-79786 I. Preparation of compound SMP-79786 TIFF2026513895000024.tif53170 Step 1: Preparation of compound SMP-79786 TIFF2026513895000025.tif29170 Compound 6-maleimidohexanoic acid (15.0 mg, 0.071 mmol) and the solvent N,N-dimethylformamide (5 mL) were added to a 25 mL flask. N,N,N',N'-tetramethyl-2-(7-azabenzotriazole)uronium hexafluorophosphate (27.0 mg, 0.071 mmol) and N,N-diisopropylethylamine (11.4 mg, 0.088 mmol) were added sequentially, and the mixture was reacted at room temperature for 5 minutes. Then, intermediate A (62.0 mg, 0.059 mmol) was added, and the mixture was reacted at room temperature for 25 minutes. After confirming the completion of the reaction by TLC, the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain 48.0 mg of compound SMP-79786 (yield 65.5%). MS (ESI) m / z: 1241 [M+H] + .

[0059] II. Preparation of SMP-79786-X Coupling Method: Antibody-drug conjugates were prepared by drug coupling utilizing the disulfide bonds of the antibody. First, the antibody was prepared in a solution to a concentration of 20 mg / mL. 0.1 mL of the antibody solution was transferred to a 1.5 mL centrifuge tube, PBS (pH 7.4) / DTPA solution (293 μL) was added, followed by 5 mM TCEP aqueous solution (16.0 μL, 6.0 equivalents per antibody molecule). The mixture was incubated at 25°C for 2 hours to reduce the disulfide bonds in the antibody hinge region to sulfhydryl groups. Subsequently, 10% medical DMSO and 10 mM payload solution (13.3 μL, 10 equivalents per antibody molecule) were added to the aforementioned solution. The mixture was reacted at 25°C under shaking conditions of 400 rpm for 2 hours to link the drug-linking construct to the antibody, thereby obtaining the antibody-drug conjugate.

[0060] The antibody-drug conjugate obtained by applying the aforementioned coupling method to an anti-HER2 antibody using compound SMP-79786 as a drug-linking constructor was named SMP-79786-X.

[0061] Concentration of antibody-drug conjugate: The obtained antibody-drug conjugate solution was transferred to a 5000K (Millipore) ultrafiltration tube and centrifuged at 2°C and 3500G for 10 minutes using a high-speed cryogenic centrifuge (GENESPEED 1580R).

[0062] The sequence of the anti-HER2 antibody used in this comparative example is as follows: Inetetamab Heavy chain (SEQ ID NO:1): EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRYADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSSASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPG; Light chain (SEQ ID NO:2): DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPSRFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC.

[0063] The following examples demonstrate the beneficial effects of the present invention.

[0064] Experimental Example 1: Quality Evaluation of Antibody-Drug Conjugates 1. Experimental Method High-performance liquid chromatography (SE-HPLC) was used to detect antibody-drug conjugate aggregates (high molecular weight components). A Biocore SEC column (7.8 × 300 mm, 5 μm) was used, with a mobile phase of a mixed solution of 50 mM phosphate buffer containing 300 mM sodium chloride (pH 6.8) and isopropanol (95:5, V / V). The flow rate was 0.5 mL / min, the injection sample volume was 50 μg, and the injection volume was 10 μL. UV absorbance at 280 nm was measured for more than 30 minutes, and the antibody-drug conjugate aggregate content was calculated by the area percentage method.

[0065] The drug-to-antibody ratio (DAR) of antibody-drug conjugates was measured using reversed-phase high-performance liquid chromatography-mass spectrometry (RP-HPLC-MS). ADC samples were diluted to 1 mg / ml with 50 mmol / L Tris buffer (pH 8.0), and a freshly prepared dithiothreitol (DTT) stock solution was added to achieve a final DTT concentration of 50 mmol / L. The mixture was incubated at 37°C for 10 minutes. A PLRP-S column (1000 Å, 2.1 × 50 mm, 5 μm) was used, with a column temperature of 70°C. The flow rate was 0.25 mL / min. The injection volume was 10–20 μg. Mobile phase A was an aqueous solution containing 0.1% formic acid (V / V) and 0.025% trifluoroacetic acid (V / V), and mobile phase B was an acetonitrile solution containing 0.1% formic acid (V / V) and 0.025% trifluoroacetic acid (V / V). The gradient conditions were as follows: mobile phase B was held at 27% for 3 minutes from 0 to 3 minutes, then a linear gradient was applied from 27% to 49% mobile phase B from 3 to 25 minutes, then a linear gradient was applied from 49% to 95% mobile phase B from 25 to 26 minutes, then mobile phase B was held at 95% from 26 to 31 minutes, then a linear gradient was applied from 95% to 27% mobile phase B from 30 to 31.5 minutes, and finally mobile phase B was held at 27% from 31.5 to 45 minutes. The UV absorbance at 280 nm was measured. The mass spectrometer was set to a dry gas temperature of 350°C, a dry gas flow rate of 7.0 L / min, a nebulizer pressure of 40 psi, and a capillary voltage of +3000V in cation mode and -3000V in anion mode. The mass range was 500 to 1600 m / z, and the molecular weight (range 20000 to 70000) of each mass spectral peak was calculated by deconvolution. After calculating the drug payload of each peak by comparing it with the molecular weights of the light and heavy chains of the unbound antibody, the corresponding UV absorption peak loading was determined. The UV absorption peak area percentage of each heavy and light chain was calculated by integral calculation, and the weighted average DAR of the antibody-drug conjugate was calculated by combining it with the drug loading distribution of each peak.

[0066] The naked antibody content in antibody-drug conjugates was measured using hydrophobic interaction chromatography (HIC). A Biocore HIC-Butyl column (4.6 × 150 mm, 5 μm) was used. Mobile phase A was 100 mM phosphate buffer containing 2 M ammonium sulfate (pH 7.0), and mobile phase B was a mixture of 100 mM phosphate buffer (pH 7.0) and isopropanol (80:20, V / V). The injection volume was 50 μg, the injection volume was 10 μL, and the flow rate was 1 mL / min. Mobile phase A was held at 100% for 3 minutes. Mobile phase B was linearly gradient-transformed from 0% to 100% over 3 to 25 minutes, and then rapidly decreased to 0% over 25.1 minutes, holding this state for up to 30 minutes. UV absorbance at 280 nm was measured. The retention time of the naked antibody peak corresponding to the antibody portion of the antibody-drug conjugate was identified, and the naked antibody content in the antibody-drug conjugate was calculated based on the area percentage method.

[0067] 2. Experimental Results [Table 1]

[0068] SMP-656 contains the same antibodies and low-molecular-weight toxins as SMP-79786-X; the only difference is in the linker design of SMP-656. The only difference is the introduction of the TIFF2026513895000027.tif21170 structure. However, as can be seen from the table above, SMP-656 showed lower aggregate and uncoated antibody content compared to SMP-79786-X under conditions where the DAR values ​​were similar. Furthermore, under the same coupling conditions, the measured DAR of SMP-656 was closer to the set DAR value.

[0069] Therefore, the experimental results described above demonstrate that the ADC of the present invention has low aggregate content and naked antibody content, and its DAR value is in close agreement with the designed DAR value. As a result, improved stability of the ADC, reduced immunogenicity, suppression of competitive inhibition, and improved drug therapeutic effect can be expected.

[0070] Experimental Example 2: Plasma Stability Test of Antibody-Drug Conjugates 1. Experimental Method (1) Sample preparation: 980 μL each of blank plasma (derived from mouse, monkey, dog, and human, anticoagulated with heparin sodium) was pre-incubated at 37°C for 5 minutes, then 20 μL of ADC sample was added to adjust the ADC concentration in the plasma to approximately 100 μg / mL (two samples were prepared in duplicate). 50 μL of plasma sample was collected at each predetermined time point, 150 μL of ice-cold acetonitrile containing an internal standard was added, and the mixture was vortexed. Then, 500 μL of extraction solvent (methyl tert-butyl ether + ethyl acetate = 50 + 50) was added and the mixture was vortexed again. After centrifugation (12000 rpm, 10 minutes), 600 μL of the supernatant was collected, dried under a nitrogen stream, redissolved in 100 μL of acetonitrile / water (50:50), and stored at -80°C. After all time point samples had been collected, the analysis was performed collectively.

[0071] (2) Blank sample: After incubating each blank plasma matrix at 37°C, take 50 μL, add ice-cold acetonitrile without internal standard material, and process according to the sample preparation method to prepare a blank sample. (3) Zero sample: After incubating each blank plasma matrix at 37°C, 50 μL is taken, ice-cold acetonitrile containing an internal standard is added, and the sample is processed according to the sample preparation method to prepare a zero sample. (4) Calibration standard samples and quality control (QC) samples: Diluted standard solutions of calibration standard samples (containing 20 ng / mL to 40 μg / mL of the low molecular weight toxin to be measured) and diluted standard solutions of QC samples (containing high, medium, and low concentrations of the low molecular weight toxin, prepared in duplicate) were each added to pre-incubated plasma. Then, 50 μL was taken and ice-cold acetonitrile containing the internal standard was added. Calibration standard samples and QC samples were prepared by processing according to the sample preparation method. (5) The chromatography conditions were as follows: Column: Waters Xbridge C18 (4.6 × 50 mm, 3.5 μm), Mobile phase A: 0.1% formic acid aqueous solution, Mobile phase B: 0.1% formic acid acetonitrile solution. Gradient: From 0 to 2.3 minutes, mobile phase A was changed from 95% to 5%, held for 1 minute, and then from 3.3 to 3.4 minutes, mobile phase A was changed from 5% to 95%, held for 0.4 minutes. Analysis was performed at a column temperature of 40°C, a flow rate of 2 mL / min, and an injection volume of 10 μL.

[0072] (6) Mass spectrometry conditions: positive ion SIM mode. Target low molecular weight toxin m / z 730.5 (collision-induced dissociation energy: 90), internal standard m / z 787.4 (collision-induced dissociation energy: 120). Peak width 0.02 min, dry gas flow rate 9 L / min, nebulizer pressure 60 psi, nebulizer chamber temperature 350 °C.

[0073] (7) Data processing: Linear regression analysis (weighting coefficient: 1 / X) was performed with the concentration of the measured substance in the calibration curve on the x-axis and the peak area ratio of the measured compound and the internal standard substance on the y-axis. 2 The following was performed: The concentration of the target substance in each sample solution was calculated, and the percentage of the measured value relative to the theoretical content (calculated based on DAR value and ADC concentration) was defined as the payload release ratio.

[0074] 2. Experimental Results Figure 1 confirms that SMP-656 exhibits significantly higher plasma stability compared to DS-8201a in mouse, dog, monkey, and human plasma.

[0075] Experimental Example 3: In vivo efficacy study of antibody-drug conjugates 1. Experimental Method 1.1 Construction of a tumor model: Tumor cells from ovarian cancer (SKOV-3), gastric cancer (NCI-N87), breast cancer (JIMT-1), pancreatic cancer (Capan-1), and breast cancer (HCC1954) were cultured in their respective culture media and maintained in an incubator at 37°C and saturated humidity with 5% CO2. Tumor cells in the logarithmic growth phase were harvested, resuspended in basal medium, and the cell concentration was increased to 8 × 10⁶. 7 The cells were adjusted to a concentration of cells / mL. Under sterile conditions, 0.1 mL of cell suspension was administered (inoculation concentration: 8 × 10⁶). 6 (0.1 mL of cells per mouse) was inoculated subcutaneously into the right axilla of the mouse.

[0076] 1.2 Grouping and Observation of Administration Tumor volume is approximately 100-200 mm 3 Upon reaching a certain stage, the animals were randomly divided into groups based on tumor volume, and the day of group division was recorded as Day 0. Administration was carried out based on animal body weight (ADC dosage is shown in Figure 2). During the study period, animal body weight and tumor volume were measured twice a week, and clinical symptoms were observed and recorded daily. The study was conducted for 21 days, and at the end of the study, after the final body weight measurement, the remaining animals were euthanized with CO2, and the tumors were excised, weighed, and photographically recorded.

[0077] 2. Experimental Results As shown in Figure 2, in tumor models of ovarian cancer SKOV-3, gastric cancer NCI-N87, pancreatic cancer Capan-1, breast cancer JIMT-1, and breast cancer HCC1954, SMP-656 showed significantly higher tumor suppressive effects compared to DS-8201a under the same dosage (1 mg / kg) conditions, confirming that the in vivo antitumor effect of SMP-656 is clearly superior to that of DS-8201a.

[0078] The experimental results described above demonstrate that the ADC of the present invention exhibits a significantly improved in vivo antitumor effect.

[0079] Experimental Example 4: Evaluation of the duration of action of antibody-drug conjugates in vivo. 1. Experimental Method 1.1 Construction of a tumor model: Gastric cancer NCI-N87 tumor cells were cultured in culture medium and maintained in a saturated humidity incubator at 37°C with 5% CO2. Tumor cells in the logarithmic growth phase were harvested, resuspended in basal medium, and the cell concentration was increased to 1 × 10⁶. 8 The cells were adjusted to a concentration of cells / mL. Under sterile conditions, 0.1 mL of cell suspension was administered (inoculation concentration: 1 × 10⁶). 7 (0.1 mL of cells per mouse) was inoculated subcutaneously into the right axilla of the mouse.

[0080] 1.2 Grouping and Observation of Administration The tumor volume is approximately 110 mm. 3 Upon reaching a certain stage, the animals were randomly divided into groups based on tumor volume, and the day of group division was recorded as Day 0. Administration was carried out based on animal body weight (dosage in terms of low molecular weight toxin is shown in Figure 3). During the study period, animal body weight and tumor volume were measured twice a week, and clinical symptoms were observed and recorded daily. The study was conducted for 62 days, and at the end of the study, after the final body weight measurement, the remaining animals were euthanized with CO2, and the tumors were excised, weighed, and photographically recorded.

[0081] 2. Experimental Results As shown in the experimental results in Figure 3, under conditions where the dose and frequency of low molecular weight toxin administration were the same, the linker... SMP-656 incorporating TIFF2026513895000028.tif21170 was confirmed to suppress tumor growth sustainably over a longer period without causing tumor regrowth. On the other hand, DS-8201a and SMP-79786-X, which do not incorporate this structure into the linker, showed tumor regrowth starting 20 days after administration.

[0082] The results described above demonstrate that the ADC of the present invention significantly improves the effect of sustained tumor suppression in vivo and effectively suppresses tumor regrowth.

[0083] Experimental Example 5: Safety Evaluation of Antibody-Drug Conjugates 1. Experimental Method This study established four groups: a solvent control group and groups receiving different doses of SMP-656 (5 mg / kg, 10 mg / kg, and 15 mg / kg). Each group consisted of 10 animals (5 females and 5 males). The monkeys in each group were administered SMP-656 intravenously at doses of 5, 10, and 15 mg / kg in a volume of 10 mL / kg (infusion over 30 minutes) for a total of three doses, once every three weeks (a total of nine weeks), followed by a four-week recovery period.

[0084] During the study period, detailed clinical observations, weight measurements, and food intake monitoring were performed once a week. Clinicopathological examinations (hematology, coagulation, serum biochemistry, urinalysis) were performed on Day 7, Day 50, and Day 71. Irritation at the injection site was observed once a week during the administration period (2-4 hours after administration on the day of administration), and once a week during the recovery period. For animals showing symptoms, the frequency of observation was increased to daily until the symptoms disappeared. All surviving animals underwent toxicological studies in parallel on Day 1 and Day 22. At the end of the administration period and the end of the recovery period, all surviving animals were euthanized according to the plan, autopsies and macroscopic examinations were performed, and tissues and organs were collected for weight measurement or fixation and preservation (for pathological examination).

[0085] 2. Experimental Results [Table 2]

[0086] Both MORAb-202 and SMP-656 are ADCs that use eribulin as a low-molecular-weight toxin and have a set DAR value of 4. As shown in Table 2, SMP-656 showed a higher HNSTD (maximum dose without serious toxicity), and when compared in eribulin equivalent, it improved the HNSTD of eribulin by 6.7 times. This demonstrates the superior safety of SMP-656.

[0087] Based on the above, the present invention provides an antibody-drug conjugate, a method for preparing the same, and its use. The antibody-drug conjugate has a low aggregate content and a low percentage of unadulterated antibody, an appropriate DAR value, exhibits excellent plasma stability and antitumor activity, and can effectively suppress tumor regrowth, making it highly promising for clinical application.

Claims

1. An antibody-drug conjugate, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof, wherein the antibody-drug conjugate is as shown in formula I: In the formula, Ab is an anti-HER2 antibody, and the anti-HER2 antibody is either Inetetamab or trastuzumab. q is an integer between 1 and 20. An antibody-drug conjugate characterized in that D is the drug portion, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof.

2. D is a cytotoxic drug, a treatment for autoimmune toxicity, or an anti-inflammatory drug. Preferably, D is a drug that targets DNA or a drug that targets tubulin. More preferably, D is The antibody-drug conjugate according to claim 1, characterized in that it is one of the compounds or derivatives thereof, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof.

3. The antibody-drug conjugate has the structure shown in Formula II: In the formula, Ab is an anti-HER2 antibody, and the anti-HER2 antibody is either Inetetamab or trastuzumab. The antibody-drug conjugate according to claim 2, characterized in that q is an integer from 1 to 20, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof.

4. The antibody-drug conjugate according to claim 1, characterized in that the heavy chain sequence of Inetetamab is as shown in SEQ ID NO: 1, and the light chain sequence is as shown in SEQ ID NO: 2, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof.

5. The antibody-drug conjugate according to claim 1, characterized in that the heavy chain sequence of trastuzumab is as shown in SEQ ID NO: 3, and the light chain sequence is as shown in SEQ ID NO:

4. The stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated thereof.

6. The antibody-drug conjugate according to claim 1, characterized in that the DAR value of the antibody-drug conjugate is 1.00 to 20.00, preferably 2.0 to 8.0, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof.

7. The antibody-drug conjugate according to claim 6, characterized in that the DAR value of the antibody-drug conjugate is 2.0 to 5.0, preferably 4.08 ± 0.

5.

8. A pharmaceutical preparation for the prevention and / or treatment of tumors, characterized in that it is a preparation prepared by adding an antibody-drug conjugate as described in claim 1, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof, as an active ingredient, and a pharmaceutically acceptable excipient.

9. The pharmaceutical preparation according to claim 8, characterized in that the preparation is an oral preparation or an injectable preparation.

10. Use of the antibody-drug conjugate described in claim 1, or its stereoisomer, or its optical isomer, or its salt, or its deuterated form, in the preparation of a pharmaceutical formulation for the prevention and / or treatment of tumors.

11. The use according to claim 10, characterized in that the tumor is selected from lung cancer, urethral cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, stomach cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma, and bile duct tumor.

12. A method for preparing an antibody-drug conjugate, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof, the method comprising the step of obtaining an antibody-drug conjugate by coupling an anti-HER2 antibody with compound III, wherein the structure of compound III is as follows: A method for preparing an antibody-drug conjugate according to claim 1, or a stereoisomer thereof, or an optical isomer thereof, or a salt thereof, or a deuterated thereof, characterized in that D is the drug portion according to claim 1.

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