Targeted b7-h3 antibody-mmae conjugates and uses thereof

Abstract

The application relates to the fields of biopharmaceuticals and biotechnology, in particular to a B7-H3 antibody-MMAE conjugate and application thereof. The application provides an antibody-drug conjugate, which comprises a compound shown in formula I or a pharmaceutically acceptable salt or solvate thereof: the application provides the use of the prepared antibody-drug conjugate in treating high-proliferation diseases or lesions, in particular in the treatment of brain glioma.

Description

Technical Field

[0001] This application relates to the fields of biopharmaceuticals and biotechnology, and in particular to antibody-MMAE conjugates targeting B7-H3 and their applications. Background Technology

[0002] B7-H3 (CD276) is a transmembrane protein that plays a crucial role in angiogenesis and metastasis, tumor migration and invasion, and tumor growth. Studies have shown that B7-H3 is highly expressed in various solid tumors, including glioma, non-small cell lung cancer, renal cell carcinoma, breast cancer, and prostate cancer, while its expression is low in normal tissues. Therefore, B7-H3 is a suitable target for anticancer drug development.

[0003] Antibody-drug conjugates (ADCs) consist of three parts: an antibody, a linker, and a highly active drug molecule. Through the targeting action of the antibody, the highly active drug molecule can be specifically delivered to the tumor site, thus achieving a highly effective and low-toxicity effect, and can be used for targeted cancer therapy. Currently, 14 ADCs have been approved by the FDA, and more than 100 other ADCs are in clinical trials. Drug development based on B7-H3 has employed various methods, including antibody drugs, bispecific antibodies, CAR-T therapy, and small molecule drugs, but to date, there are no marketed drugs specifically targeting this target. ADCs targeting B7-H3 are gradually gaining popularity. Summary of the Invention

[0004] To address the technical problems in the prior art, the purpose of this application is to provide a B7-H3-targeting antibody-matrix agonist (ADC) and its applications. This invention provides a method for preparing a uniform B7-H3-targeting antibody-drug conjugate (ADC) via bridging and its application in targeted cancer therapy, particularly in the treatment of gliomas.

[0005] To achieve the above and other related objectives, this application provides an antibody-drug conjugate comprising a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof:

[0006]

[0007] Formula I

[0008] Wherein, Anti-B7-H3 is an anti-B7-H3 antibody, D is the drug, L is the linker group; n represents the drug-to-antibody ratio (DAR), where n is a natural number from 1 to 5.

[0009] In the specific implementation of this application, the antibody-drug conjugate is selected from SS-401, and its structural formula is:

[0010] .

[0011] The Anti-B7-H3 antibody 401 mentioned in this application refers to Anti-B7-H3 antibody hu4G4 in patent 202210020231.3, and will be referred to as Anti-B7-H3 antibody 401 below.

[0012] This application also provides a method for preparing an antibody-drug conjugate, comprising the following steps:

[0013] 1) Compound 1, 11-azido-3,6,9-trioxoundecanoic acid, N-methylmorpholine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 1-hydroxybenzotriazole were reacted in solution to give compound 2:

[0014] ;

[0015] 2) Compound 3, anhydrous copper sulfate, and sodium ascorbate were added to the solution of compound 2. After the reaction, the insoluble matter was removed by filtration, and compound 4 was obtained.

[0016]

[0017] 3) The B7-H3 monoclonal antibody 401 solution and tris(2-carboxyethyl)phosphine solution were subjected to a first shaking reaction, followed by the addition of compound 4 solution for a second shaking reaction to remove small molecule compounds, yielding the antibody-drug conjugate SS-401:

[0018] .

[0019] This application also provides the use of the aforementioned antibody-drug conjugate in the preparation of cancer-targeted therapy products or products for the treatment of proliferative diseases.

[0020] Compared with the prior art, the beneficial effects of this application are as follows:

[0021] 1. This invention is based on bis(ethylenesulfonamide) linker technology and antibody-drug conjugates that specifically target B7-H3.

[0022] 2. This invention is based on the breaking of the intrinsic sulfur-sulfur bonds of the antibody, followed by bridging with the drug via a diethylene sulfonamide linker, which can yield a homogeneous conjugate product with a DAR of around 4.

[0023] 3. The antibody-drug conjugate provided by this invention has excellent effects in vivo and in vitro.

[0024] 4. This invention provides the use of the prepared antibody-drug conjugate in the treatment of highly proliferative diseases or lesions, particularly in the treatment of glioma. Attached Figure Description

[0025] Figure 1 This invention presents a typical preparation route and structure of drug-linker involved in the present invention.

[0026] Figure 2 The image shows the HR-ESI-MS spectrum of the ADC SS-401 involved in this invention.

[0027] Figure 3 This is a diagram showing the expression of B7-H3 in cells U251, U87, and U87 / B7-H3 KO involved in this invention.

[0028] Figure 4 This invention relates to the in vitro inhibitory effects of ADC SS-401, monoclonal antibody 401, and MMAE on the proliferation of U251 cells.

[0029] Figure 5 This invention relates to the in vitro inhibitory effects of ADC SS-401, monoclonal antibody 401, and MMAE on the proliferation of U87 cells.

[0030] Figure 6 This invention relates to the in vitro inhibitory effects of ADC SS-401, monoclonal antibody 401, and MMAE on the proliferation of U87 / B7-H3 KO cells.

[0031] Figure 7 The affinity of the ADC SS-401 and monoclonal antibody 401 involved in this invention for U87 cells at different concentration gradients is shown.

[0032] Figure 8 This refers to the endocytosis effect of ADC SS-401 and monoclonal antibody 401 in U87 cells involved in this invention.

[0033] Figure 9 This is a live imaging image of the fluorescently labeled conjugate Cy5.5-SS-401 involved in this invention in a glioma xenograft tumor.

[0034] Figure 10 The images show the tumor sites, heart, spleen, liver, stomach, and kidneys of mice in xenograft gliomas, using the fluorescently labeled conjugate Cy5.5-SS-401 involved in this invention.

[0035] Figure 11 This invention relates to the inhibitory effect of ADC SS-401 and monoclonal antibody 401 on U87 glioma xenograft tumors.

[0036] Figure 12 The effects of the ADC SS-401 and monoclonal antibody 401 involved in this invention on the body weight of mice with U87 glioma xenografts.

[0037] Figure 13 This is a structural diagram of the antibody-drug conjugate SS-401 of the present invention. Detailed Implementation

[0038] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0039] The inventors of this application, through extensive research and exploration, discovered the B7-H3 antibody-MMAE conjugate and its applications, and completed this application based on this discovery.

[0040] This application provides an antibody-drug conjugate comprising a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof:

[0041]

[0042] Formula I

[0043] In this invention, Anti-B7-H3 is an anti-B7-H3 antibody, D is the drug, L is the linker group, and n represents the drug-to-antibody ratio (DAR), where n is a natural number from 1 to 5. The drug-to-antibody ratio (DAR) refers to the average number of payload molecules attached to a single monoclonal antibody, typically between 2 and 4 molecules. This invention is based on bis(ethylenesulfonamide) linker technology and the synthesis of antibodies specifically targeting B7-H3, resulting in antibody-drug conjugates targeting B7-H3.

[0044] In this invention, the term "pharmaceutically acceptable salt" generally refers to any salt that is physiologically tolerable when used in a suitable manner for treatment (particularly when applied or used in humans and / or mammals). This generally means that it is non-toxic, particularly as a result of an anti-ion. These physiologically acceptable salts can be formed with cations or bases, and in the context of this invention, particularly when administered in humans and / or mammals, they should be understood as salts formed from at least one compound provided according to this invention, typically an acid (deprotonated), such as an anion, and at least one physiologically tolerable cation (preferably an inorganic cation). Specifically, in the context of this invention, this may include salts formed with alkali metals and alkaline earth metals, as well as salts formed with ammonium cations (NH4+). +The salts formed can specifically include, but are not limited to, salts formed with (mono) or (di) sodium, (mono) or (di) potassium, magnesium, or calcium. These physiologically acceptable salts can also be formed with anions or acids, and in the context of this invention, particularly when administered to humans and / or mammals, they should be understood as salts formed by at least one compound provided according to this invention, typically protonated (e.g., on nitrogen), such as a cation, and at least one physiologically tolerable anion. In the context of this invention, salts can specifically include salts formed with physiologically tolerable acids, i.e., salts formed by specific active compounds with physiologically tolerable organic or inorganic acids, specifically including, but not limited to, salts formed with hydrochloric acid, hydrobromic acid, hydroiodic acid; sulfuric acid, persulfate, pyrosulfonic acid; phosphoric acid, nitric acid; methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid; formic acid, acetic acid, acetoacetic acid, trifluoroacetic acid, pyruvic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid; benzoic acid, salicylic acid, 2- Salts formed from (4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphtholic acid, nicotinic acid, pectic acid, 3-phenylpropionic acid, picric acid, terpentine; 2-hydroxyethanesulfonic acid, aminosulfonic acid, dodecyl sulfate, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid; citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, citric acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, aspartic acid, and sulfosalicylic acid.

[0045] In this document, the term "solvent" refers to a compound of the present disclosure or a pharmaceutically acceptable salt thereof, contained in a stoichiometric or non-stoichiometric solvent in which non-covalent molecules are bound together by force. Preferred solvents are volatile and non-toxic, and can be administered to humans in very small doses. Examples of solvents, but not limited to, include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecules are water.

[0046] In the antibody-drug conjugate provided in this application, L is selected from one or more combinations of carboxyl, amino, alkynyl, and azide groups.

[0047] In the antibody-drug conjugate provided in this application, n is 4.

[0048] In the antibody-drug conjugate provided in this application, Anti-B7-H3 is linked to the drug via an intrinsic disulfide bond through a linker group. The anti-B7-H3 antibody is an anti-B7-H3 monoclonal antibody. In a specific embodiment of this application, Anti-B7-H3 is 401 (Patent 202210020231.3).

[0049] The antibody-drug conjugates provided in this application use drugs selected from methylaurestatin E (MMAE), methylaurestatin F (MMAF), compressoritine A, cytarabine, gemcitabine, maytansine, DM1, DM4, chachiin and its derivatives, doxorubicin, pyrrolobenzodiazepines and their derivatives, SN-38, camptothecin and its analogues, α-ammantin, and tubulin lysin analogues. In a specific embodiment of this application, the drug is selected from MMAE.

[0050] The antibody-drug conjugate provided in this application has the following structural formula:

[0051] .

[0052] It can also be expressed as:

[0053] It can also be expressed as Figure 13 The structure shown.

[0054] This application also provides a method for preparing the aforementioned antibody-drug conjugate, comprising the following steps:

[0055] 1) Compound 1, 11-azido-3,6,9-trioxoundecanoic acid, N-methylmorpholine (NMM), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCl), and 1-hydroxybenzotriazole (HOBT) were reacted in solution to give compound 2:

[0056] ;

[0057] 2) Compound 3, anhydrous copper sulfate, and sodium ascorbate were added to the solution of compound 2. After the reaction, the insoluble matter was removed by filtration, and compound 4 was obtained.

[0058]

[0059] 3) The B7-H3 monoclonal antibody 401 solution and tris(2-carboxyethyl)phosphine solution were subjected to a first shaking reaction, followed by the addition of compound 4 solution for a second shaking reaction to remove small molecule compounds, yielding the antibody-drug conjugate:

[0060] .

[0061] In the preparation method provided in this application, step 1) refers to reacting compound 1, 11-azido-3,6,9-trioxoundecanoic acid, NMM, EDCl, and HOBT in solution to obtain compound 2. The solvent for the solution is N,N-dimethylformamide (DMF). The reaction temperature is room temperature, and the reaction time is 2–24 h; specifically, 2–10 h, 10–12 h, 12–16 h, or 16–24 h, etc.

[0062] In the preparation method provided in this application, step 2) refers to adding compound 3, anhydrous copper sulfate, and sodium ascorbate to a solution of compound 2, reacting, filtering to remove insoluble matter, and separating to obtain compound 4. The solvent for the solution of compound 2 is tert-butanol (…). t A mixed solution of BuOH, H2O, and DMF. In a specific embodiment of this application, t The volume ratio of BuOH, H2O, and DMF is 1:1:1. The reaction temperature is room temperature, and the reaction time is 1–4 h; specifically, 1–2 h, 2–3 h, or 2–3 h, etc. Separation is achieved by HPLC. The HPLC conditions are: stationary phase: C-18 column, mobile phase: CH3CN / H2O = 10–100%, for a total of 30 minutes.

[0063] In the preparation method provided in this application, step 3) refers to subjecting B7-H3 monoclonal antibody solution 401 and tris(2-carboxyethyl)phosphine solution to a first shaking reaction, followed by adding compound 4 solution for a second shaking reaction to remove small molecule compounds, thereby obtaining the antibody-drug conjugate. Step 3) is performed in a microplate. The solvent for B7-H3 monoclonal antibody solution 401 is phosphate-buffered saline (PBS), pH=7.4. The solvent for tris(2-carboxyethyl)phosphine solution is pure water, pH=7.0. The reaction temperature for the first shaking reaction is room temperature, and the reaction time is 0.5~2h; specifically, 0.5~1h, 1~1.5h, or 1.5~2h, etc. The solvent for compound 4 solution is dimethyl sulfoxide (DMSO), and the reaction temperature for the second shaking reaction is room temperature, and the reaction time is 2~18h; specifically, 2~6h, 6~12h, or 12~18h, etc. Small molecule compounds were removed using Zeba™ SpinDesalting Columns (7K MWCO, 0.5 mL). This invention is based on the breaking of the intrinsic sulfur-sulfur bonds of the antibody, followed by bridging with the drug via a diethylene sulfonamide linker, to obtain a homogeneous conjugate product SS-401 with a DAR of approximately 4.

[0064] This application also provides the use of the aforementioned antibody-drug conjugate in the preparation of cancer-targeted therapy products or products for the treatment of proliferative diseases.

[0065] In the application provided in this application, the target of cancer targeted therapy is B7-H3. Proliferative diseases are characterized by cells producing an antigen or target that specifically binds to an antibody against B7-H3. Further, proliferative diseases are lesions expressing B7-H3. Even further, proliferative diseases are gliomas. This invention provides an antibody-drug conjugate that exhibits excellent efficacy both in vivo and in vitro, and can be applied to treat highly proliferative diseases or lesions, especially gliomas.

[0066] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0067] Example 1

[0068] Preparation of antibody-drug conjugates

[0069] Preparation route as follows Figure 1 As shown, the process is as follows:

[0070] Compound 1 (referencing Bioorganic Chemistry 134 (2023) 106463) (100 mg, 0.139 mmol), 11-azido-3,6,9-trioxoundecanoic acid (35.58 mg, 0.153 mmol), N-methylmorpholine (NMM) (140.6 mg, 1.39 mmol), EDCl (53.48 mg, 0.279 mmol), and HOBt (37.92 mg, 0.279 mmol) were dissolved in DMF (5 mL) and reacted at room temperature for 12 h. The product, a white solid 2 (111 mg), was obtained by preparative HPLC (stationary phase: C-18 silica gel column; mobile phase: CH3CN / H2O = 10–100%, 30 min).

[0071] Compound 2 (6 mg, 0.015 mmol) was dissolved in 1.5 mL of... t Compound 3 (20 mg, 0.015 mmol), anhydrous copper sulfate (2 mg, 0.015 mmol), and sodium ascorbate (4 mg, 0.022 mmol) were added to a BuOH / H2O / DMF (1 / 1 / 1) mixed solution. After reacting the mixture at room temperature for 2 hours, insoluble matter was removed by filtration, and 13 mg of white solid 4 was obtained by preparative HPLC (stationary phase: C-18 column, mobile phase: CH3CN / H2O = 10–100%, 30 min). . 1 H NMR (500 MHz, DMSO) δ10.13 - 10.06 (m, 1H), 8.44 (q, J = 6.6, 5.9 Hz,1H), 8.35 (dd, J = 17.4, 9.8 Hz, 2H), 7.61 - 7.55 (m, 2H), 7.39-7.22 (m, 8H),7.20-7.13 (m, 1H), 6.83-6.67 (m, 2H), 6.20-6.01 (m, 5H), 5.03 (ddd, J = 33.8,26.7, 13.5 Hz, 2H), 4.51-4.11 (m, 12H), 3.95 (qd, J = 18.4, 15.8, 4.9 Hz,6H), 3.76 (dt, J = 11.6, 5.5 Hz, 3H), 3.58-3.49 (m, 10H), 3.26-3.08 (m, 11H),3.07-2.82 (m, 10H), 2.68 (td, J = 11.1, 10.5, 5.3 Hz, 1H), 2.15-1.90 (m, 4H),1.83- 1.62 (m, 3H), 1.63-1.40 (m, 3H), 1.39-1.21 (m, 2H), 1.10-0.96 (m, 6H),0.90-0.71 (m, 29H). 13 C NMR (151 MHz, DMSO) δ10.3, 14.0, 15.0, 15.3, 15.5,15.9, 17.9, 18.0, 18.5, 19.2, 22.1, 23.1, 24.3, 25.3, 26.8, 28.7, 29.0, 29.3,29.9, 31.1, 31.3, 33.3, 34.5, 40.4, 41.8, 43.2, 43.7, 44.4, 44.6, 45.0, 46.2,47.1, 47.2, 47.5, 49.2, 49.4, 49.8, 53.2, 54.1, 54.2, 54.4, 56.6, 57.2, 58.2,58.7, 60.3, 60.9, 67.9, 68.7, 69.6, 69.6, 69.7, 70.3, 74.8, 81.6, 85.4,119.0, 123.0, 126.4, 126.5, 126.7, 126.7, 127.4, 127.6, 127.7, 127.8, 128.2,129.7, 129.8, 132.6, 132.7, 135.6, 135.6, 143.7, 144.5, 158.9, 168.0, 168.1,168.8, 169.0, 170.5, 170.7, 172.4. ESI-HRMS calculated for C 78 H 124 N 16 O 21 S2 [M+H] + : 1684.8568, Found: 1684.8512.

[0072] Compound 4 was dissolved in DMSO to prepare a 10 mmol / L solution; monoclonal antibody 401 was dissolved in PBS (25 mmol / L Tris, pH 7.4) buffer to prepare a 3 mg / mL solution; TCEP was dissolved in water to prepare a 10 mM solution, and the pH was adjusted to 7.0 with NaOH / H3PO4. 100 μL of monoclonal antibody solution and 0.83 μL of TCEP solution were added to a 330 μL 96-well plate, and the plate was incubated at room temperature for 1 h on a microplate shaker. Then, 1.67 μL of the prepared compound 4 was added, and the plate was incubated at room temperature for 12 hours on a microplate shaker. Excess small molecule compounds were removed by passing the solution through a Zeba™ Spin Desalting Column (7K MWCO, 0.5 mL). The corresponding antibody-drug conjugate (ADC) (SS-401) was obtained. The reaction results were analyzed by UPLC-MS (ABsciex 4600).

[0073] Example 2

[0074] Detection of B7-H3 antigen expression in cells

[0075] Prepare 500 mL FACS buffer (PBS + 0.1% BSA). Collect U87 (human glioma cells), U87-B7-H3-KO (B7-H3 gene knocked out in U87 cells using conventional techniques), and U251 (human glioma cells) cells into 15 mL centrifuge tubes. Wash with FACS buffer, resuspend the cells, and aliquot them into 1.5 mL centrifuge tubes (ensuring an adequate number of viable cells per tube and triplicate for each cell type). Centrifuge again to collect the cell pellet. Then, incubate all cells on ice for 30 min with 200 μL of 100 nM FITC anti-Human CD276 (B7-H3) solution. After incubation, wash twice with FACS buffer. Resuspend each sample in 200 μL of 1 μg / mL DAPI solution. Measure the fluorescence intensity of FITC using a CytoFLEX FlowCytometer. Analyze and process the data using FlowJo 10 software.

[0076] The results are as follows Figure 3 As shown.

[0077] Example 3

[0078] Cell proliferation inhibition detection

[0079] Cells in the logarithmic growth phase were seeded into 96-well plates with 100 µL of culture medium per well. For suspension cells, 2 × 10⁶ cells were seeded per well. 4For individual cells, after plating, medication can be added directly and incubated for 48 hours before processing; for adherent cells, 3 × 10⁶ cells per well are recommended. 3 Each cell line needs to be incubated in an incubator for 24 hours to allow for complete cell adhesion before adding the drug. The ADC (SS-401) prepared in Example 1, monoclonal antibody 401, and the small molecule toxic substance MMAE (Bide Pharmaceuticals) were diluted in a specific concentration gradient and added to cell plates for incubation for 72 hours. Three replicates were set up for each concentration. A solvent control group (cells only, no drug added) and a blank control group (only an equal volume of culture medium added) were also set up. After drug treatment, viability of cells was detected using the CCK-8 reagent, and the absorbance (OD value) at 450 nm was read using a microplate reader. The inhibition rate (%) was calculated using the formula: = (OD value / (Cells only, but no drug added)). 对照 -OD 给药 ) / (OD 对照 -OD 空白 ×100%. Note: The cell seeding concentration and CCK8 staining time need to be determined through trial and error. Finally, ensure that the OD value is between 1.0 and 1.5 during the staining time.

[0080] The results are as follows Figures 4-6 As shown.

[0081] Example 4

[0082] Affinity assay of antibody-drug conjugates

[0083] During this experiment, all samples must be centrifuged and incubated at 4°C (centrifuged in a low-temperature centrifuge or incubated on ice).

[0084] 1. Cell Collection: U87 cells in good growth condition were collected by trypsin digestion and transferred to 15 mL centrifuge tubes. The cells were washed with FACS buffer, centrifuged again, resuspended, and counted. Cells were then centrifuged at 5 × 10⁻⁶ cells per tube. 5 Aliquot the live cells into 1.5 mL centrifuge tubes, centrifuge at 2000 rpm for 3 min, and discard the supernatant;

[0085] 2. Antibody / ADCs binding to cells: Dilute monoclonal antibody 401 or ADC (SS-401) with PBS solution, and resuspend the cells in 200 μL of monoclonal antibody 401 or SS-401 solution of different concentrations (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300 nM) to form a cell suspension. Incubate on ice for 30 min. During incubation, the cells need to be repeatedly blown and aspirated to prevent them from settling to the bottom.

[0086] 3. Washing: Wash cells twice with FACS buffer;

[0087] 4. Incubation with secondary antibody: Add 200 μL of GoatAnti-Human IgG H&L (DyLight® 650) diluted at a ratio of 1:200 to each sample and incubate on ice in the dark for 30 min.

[0088] 4. Washing: Wash cells twice with FACS buffer;

[0089] 6. DAPI staining and flow cytometry: Resuspend in DAPI solution (concentration of 1 μg / mL) and perform flow cytometry analysis after 15 minutes.

[0090] The results are as follows Figure 7 As shown.

[0091] Example 5

[0092] Antibody-drug conjugate endocytosis assay

[0093] During this experiment, all 4℃ control group samples must be centrifuged and incubated at 4℃ (centrifuged in a low-temperature centrifuge or incubated on ice).

[0094] 1. Cell Collection: Collect well-grown U87 cells by trypsin digestion according to the adherent cell treatment method and transfer them to 15 mL centrifuge tubes. Wash with FACS buffer; centrifuge the cells again, resuspend and count, and then centrifuge at 5 × 10⁻⁶ cells per centrifuge tube. 5 Each live cell was aliquoted into 1.5 mL centrifuge tubes, centrifuged at 2000 rpm for 3 min, and the supernatant was discarded. A 4℃ control group and a 37℃ experimental group were set up.

[0095] 2. Antibody / ADCs binding to cells: Dilute monoclonal antibody 401 or SS-401 with PBS solution, resuspend the cells in 200 μL of 100 nM monoclonal antibody 401 or SS-401 solution to form a cell suspension, and incubate on ice for 30 min. During incubation, the cells need to be blown and aspirated multiple times to prevent the cells from settling to the bottom.

[0096] 3. Washing: Wash cells twice with FACS buffer;

[0097] 4. Internalization process: After resuspending the cells in 200 μL of fresh complete culture medium in each centrifuge tube, the 37℃ group was transferred to a 37℃ constant temperature incubator and incubated for 3 hours to allow the monoclonal antibody 401 or SS-401 bound to the cell surface to be internalized; the 4℃ group was incubated on ice for the same time as a control.

[0098] 5. Termination of internalization: Transfer the 37℃ group to ice and add 200 μL of cold PBS solution to terminate internalization. At the same time, add an equal volume of cold PBS to the 4℃ group, centrifuge at 2000 rpm at 4℃ for 3 min, discard the supernatant, and wash once with FACS buffer;

[0099] 6. Incubation with secondary antibody: Add 200 μL of 1:200 diluted secondary antibody Goat Anti-Human IgG H&L (DyLight® 650) to each sample and incubate on ice in the dark for 30 min.

[0100] 7. Washing cells for unbound secondary antibody: Wash cells twice with FACS buffer.

[0101] 8. DAPI staining and flow cytometry: Resuspend in DAPI solution (concentration of 1 μg / mL) and perform flow cytometry analysis after 15 minutes.

[0102] Internalization efficiency calculation formula: Internalization efficiency (%) = (fluorescence intensity of 4℃ group - fluorescence intensity of 37℃ group) / fluorescence intensity of 4℃ group × 100%.

[0103] The results are shown in Figure 8.

[0104] Example 6

[0105] Intra-absorptive efficacy assay of antibody-drug conjugates

[0106] Five-week-old male BALB / c nude mice of SPF grade were purchased and housed in the SPF-grade animal facility of the National Protein Center for one week to acclimatize. During this period, the mice's physical condition and mental state were observed. Simultaneously, U87 cells were resuscitated and expanded. When the cell number met the experimental inoculation requirements and the cells were in good growth condition, they were digested with trypsin, washed twice with PBS, resuspended, counted, and the cell density was adjusted to 1.5 × 10⁻⁶ cells using ABW matrix gel diluted 1:2. 7 / mL. Administer 3 × 10⁹ / mL subcutaneously to each mouse in the highly vascularized area above the right groin of the hind limb. 6 200 μL of cells were injected, and the cells were gently mixed by repeated blowing during injection. Tumor volume and body weight changes in mice were monitored every four days after inoculation (using calipers and an electronic balance). The formula for tumor volume is: Tumor volume (mm²) = ... 3 = Length (mm) × Width 2 (mm) 2 ×0.5. When the average tumor volume in mice was observed to be approximately 150 mm... 3Around 10:00 AM, mice meeting the tumor-forming criteria were randomly divided into groups according to experimental requirements to ensure that the average tumor volume of each group was similar. Then, tail vein injection was administered. The appropriate dosing regimen was selected according to the experimental design; in this experiment, the tail vein injection dosage was mainly 2 mg / kg. -1 and 6 mg·kg -1 The administration volume was kept at 200 μL. The first administration was recorded as day 0. The mice were administered once on day 0, 4 or 7, and administered for 2 to 3 consecutive times. During this period, the tumor volume and mouse weight changes were monitored and recorded every two days. The mice were euthanized after the experiment.

[0107] The results are as follows Figures 11-12 As shown.

[0108] Example 7

[0109] Intra-body fluorescence imaging detection of antibody-drug conjugates.

[0110] The tumor-targeting ability of ADCs and their distribution and metabolism in mice were evaluated using in vivo imaging technology in a U87 xenograft model. SS-401 was stained with Cy5.5 NHS ester. When the tumor volume of mice reached a suitable size, they were randomly divided into two groups of four mice each. SS-401 labeled with Cy5.5 NHS ester was administered at a dose of 6 mg / kg. -1 Mice were administered the drug via tail vein injection at the prescribed dose, and anesthetized at 24, 48, and 72 hours post-administration. The mice were then placed in a small animal chemiluminescence imaging system for imaging to observe changes in fluorescence signal distribution at different time points (the mouse position remained consistent during each imaging session). After 72 hours, the mice were dissected, and their heart, liver, spleen, lungs, kidneys, and tumor tissues were removed for local imaging to observe differences in organ fluorescence signal distribution. Living Image software was used for image analysis and quantification.

[0111] The results are as follows Figures 9-10 As shown.

[0112] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. An antibody-drug conjugate, characterized in that, The structural formula is: Among them, Anti-B7-H3 is an anti-B7-H3 antibody, and the anti-B7-H3 antibody is the anti-B7-H3 antibody hu4G4 of patent 202210020231.

3.

2. The method for preparing the antibody-drug conjugate as described in claim 1, characterized in that, Includes the following steps: 1) Compound 1, 11-azido-3,6,9-trioxoundecanoic acid, N-methylmorpholine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 1-hydroxybenzotriazole were reacted in solution to give compound 2: 2) Compound 3, anhydrous copper sulfate, and sodium ascorbate were added to the solution of compound 2. After the reaction, the insoluble matter was removed by filtration, and compound 4 was obtained. 3) The B7-H3 monoclonal antibody hu4G4 solution and tris(2-carboxyethyl)phosphine solution were subjected to a first shaking reaction, and then compound 4 solution was added for a second shaking reaction to remove small molecule compounds, thus obtaining antibody-drug conjugate SS-401.

3. The preparation method according to claim 2, characterized in that, In step 1), the solvent of the solution is N,N-dimethylformamide; the reaction temperature is room temperature; and the reaction time is 2-24 h.

4. The preparation method according to claim 2, characterized in that, In step 2), the solvent of the compound 2 solution is a mixed solution of N,N-dimethylformamide, tert-butanol and distilled water in a volume ratio of 1:1:1; And / or, in step 2), the reaction temperature is room temperature; And / or, in step 2), the reaction time is 1 to 4 hours.

5. The preparation method according to claim 2, characterized in that, Step 3) is performed in a microplate; the solvent for the B7-H3 monoclonal antibody hu4G4 solution is phosphate buffer solution; the solvent for the tris(2-carboxyethyl)phosphine solution is pure water; and the solvent for the compound 4 solution is dimethyl sulfoxide. And / or, in step 3), the reaction temperature of the first shaking reaction is room temperature, and the reaction time is 0.5~2 h; And / or, in step 3), the reaction temperature of the second oscillation reaction is room temperature, and the reaction time is 2~18 h; And / or, in step 3), the molecular weight of the small molecule compound is ≤7000.

6. The use of the antibody-drug conjugate as described in claim 1 in the preparation of a product for treating proliferative diseases, wherein the proliferative disease is glioma.

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