BMOA connexon sequence-based GD2-targeting antibody coupling drug, preparation method and application of BMOA connexon sequence-based GD2-targeting antibody coupling drug

Abstract

The invention provides a GD2 targeting antibody coupling drug based on a BMOA linker sequence, and a preparation method and application thereof, and belongs to the technical field of biological medicines. The antibody conjugate Hu3F8-BMOA-MMAE is obtained by taking elastase sensitive BMOA as a novel connexon and coupling the novel connexon with a GD2 target antibody (Hu3F8) through a cysteine coupling technology. The antibody conjugate shows good tumor cell killing ability for tumor cells expressing GD2, and can target tumor cells, so that the antibody is directly released outside the cells after targeted binding, the tumor volume is remarkably reduced, and the safety is high. Moreover, MMAE is released through cracking of intercellular elastinase to play a role, so that the bystander effect can be enhanced, the curative effect of ADC can be improved, the drug resistance mechanism related to ADC endocytosis and intracellular processing can be avoided, the enzyme specific cracking of Linker can be improved, and the toxic and side effects of ADC can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to an antibody conjugate drug targeting GD2 based on a BMOA linker sequence, a preparation method and application. BACKGROUND

[0002] As an innovative drug in the field of tumor treatment, antibody-drug conjugate (ADC) has realized a technical breakthrough in targeted therapy through a precise delivery mechanism. The core design concept of ADC is to couple a highly active cytotoxin to a specific antibody through a chemical linker, and to deliver the cytotoxic molecule to the tumor microenvironment through the high affinity recognition of the antibody to the antigen epitope, so as to reduce the systemic toxicity and enhance the therapeutic effect.

[0003] From the analysis of structural characteristics, ADC is composed of three functional modules: a targeting monoclonal antibody, a chemical linker and an effector molecule. Among them, the antibody assumes the navigation function, ensuring the precise positioning of the drug to the specific antigen on the surface of tumor cells; the linker serves as a dynamic control element, which needs to maintain structural stability in the blood circulation and controllably release the active ingredient at the target site; the effector molecule serves as the core killing unit, which exerts cytotoxicity through mechanisms such as interfering with microtubule function, inducing DNA damage or inhibiting RNA synthesis. The current clinically used effector molecules are mainly concentrated in the microtubule inhibitor class, including MMAE / MMAF derivatives based on sea slug toxin, and maytansine compounds DM1 / DM4. The linker technology is dominated by cleavable type, among which the enzyme-sensitive valine-citrulline linker and the chemically unstable cyclohexylcarboxylic acid (MCC) linker have become the industry standard configuration.

[0004] The current mechanism of traditional ADC is highly dependent on the antigen-mediated endocytosis process, which puts strict requirements on target selection: not only the tumor cell surface antigen needs to be highly expressed, but also needs to have high-efficiency internalization and intracellular processing ability. In view of this technical bottleneck, the new generation of ADC has realized a paradigm breakthrough through innovative linker technology. Based on the design of microenvironment-responsive cleavable linker, the effector molecule can be released directly outside the cell after targeted binding, thereby breaking through the dependence of traditional mode on internalization efficiency. This non-internalization-dependent mechanism not only broadens the range of applicable targets, but also significantly improves the drug treatment window, providing a new solution for solid tumor treatment.

[0005] Therefore, how to develop a kind of tumor cell targeting drug, so that the effector molecule can be released directly outside the cell after targeted binding is crucial for tumor treatment. SUMMARY

[0006] The purpose of the present invention is to provide an antibody-drug conjugate targeting GD2 based on a BMOA linker sequence, a preparation method and an application. The antibody conjugate can target tumor cells, so that the antibody is directly released extracellularly after targeted binding, significantly reducing tumor volume and having good tumor cell killing ability.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides an antibody conjugate Hu3F8-BMOA-MMAE, wherein the antibody conjugate Hu3F8-BMOA-MMAE uses the BMOA sequence as a linker and couples MMAE to the antibody through cysteine ​​coupling technology.

[0008] The present invention also provides a method for preparing an antibody conjugate Hu3F8-BMOA-MMAE, comprising the following steps: (1) Preparation of Fmoc-Bpa-Met(O2)-Oic-Abu-PAB: Fmoc-Bpa-Met(O2)-Oic-Abu was mixed with DCM, CH3OH, PABOH, and EEDQ, heated for reaction, dried, added with acetonitrile, and dried to obtain Fmoc-Bpa-Met(O2)-Oic-Abu-PAB; (2) Preparation of Bpa-Met(O2)-Oic-Abu-PAB: Fmoc-Bpa-Met(O2)-Oic-Abu-PAB was mixed with DMF and DEA, reacted, and dried to obtain Bpa-Met(O 2) -Oic-Abu-PAB; (3) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB: Mal-PEG8-COOH, EDCI, and chloroform were mixed evenly, and Bpa-Met(O2)-Oic-Abu-PAB was added, reacted, dried, dissolved in water and acetonitrile, and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB; (4) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP: Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB was mixed with DMF, DIEA and DNPC, reacted and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP; (5) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE: Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP was mixed with DMF, DIEA, HOBT, MMAE, reacted, and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE. (6) The antibody Hu3F8 was replaced into a buffer, TCEP was added, and stirred to react. After cooling, Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE was added, mixed, and reacted to obtain the antibody conjugate Hu3F8-BMOA-MMAE.

[0009] Preferably, in step (1), the mass-volume ratio of Fmoc-Bpa-Met(O2)-Oic-Abu, DCM, CH3OH, PABOH, and EEDQ is 200-300 mg:1-10 mL:1-5 mL:100-160 mg:200-300 mg; the heating reaction is performed at a temperature of 40°C for 20-30 h.

[0010] Preferably, in step (2), the mass-volume ratio of Fmoc-Bpa-Met(O2)-Oic-Abu-PAB, DMF, and DEA is 150-250 mg:2-8 mL:0.5-1.5 mL; the reaction is performed for 0.1-1 h.

[0011] Preferably, in step (3), the mass-volume ratio of Mal-PEG8-COOH, EDCI, chloroform, and Bpa-Met(O2)-Oic-Abu-PAB is 90-120 mg:20-40 mg:5-15 mL:85-100 mg; the reaction is performed for 1-4 h.

[0012] Preferably, in step (4), the mass-volume ratio of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB, DMF, DIEA, and DNPC is 100-150 mg:2-8 mL:2-30 mg:70-90 mg; the reaction is performed for 8-16 h.

[0013] Preferably, in step (5), the mass-volume ratio of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP, DMF, DIEA, HOBT, and MMAE is 30-50 mg:1-5 mL:10-30 mg:5-10 mg:30-40 mg; and the reaction time is 4-16 h.

[0014] Preferably, in step (6), the buffer comprises 5 mM PB and 5 mM EDTA; the mass-volume ratio of the antibody, TCEP, and MAP-BMOA-MMAE is 30-40 mg:60-70 µL:300-400 µL; the concentration of the antibody in the buffer is 6-7 mg / mL, the concentration of TCEP is 5-15 mM, and the concentration of MAP-BMOA-MMAE is 3-7 mM.

[0015] Preferably, in step (6), the stirring reaction time is 1-4 h, the cooling temperature is 0-5℃, and the mixing reaction time is 10-40 min.

[0016] The application further provides a preparation method of an antibody conjugate Hu3F8-BMOA-MMAE and application of the antibody conjugate in preparation of a cancer treatment drug.

[0017] Compared with the prior art, the application has the following beneficial effects: The application uses elastase-sensitive Mal-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE (BMOA) as a novel linker, and obtains an antibody conjugate Hu3F8-BMOA-MMAE by coupling GD2 target antibody (Hu3F8) through a cysteine coupling technology. The antibody conjugate has good tumor cell killing ability for tumor cells expressing GD2, can target tumor cells, enables the antibody to be released directly outside the cells after target binding, significantly reduces the tumor volume, and has high safety. The MMAE is released by intercellular elastase cleavage to play a role, which can enhance the bystander effect, improve the ADC efficacy, avoid the drug resistance mechanism related to endocytosis and intracellular processing of the ADC, improve the enzyme specificity cleavage of the linker, and reduce the toxic side effects of the ADC. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 Synthetic flow chart of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE of the present application; Figure 2 Structural schematic diagram of Hu3F8-BMOA-MMAE of the present application; Figure 3 HIC-HPLC result diagram of Hu3F8-BMOA-MMAE in Example 1 of the present application; Figure 4 SEC-HPLC result diagram of Hu3F8-BMOA-MMAE in Example 1 of the present application; Figure 5 Average tumor volume diagram of mice in Test Example 1 of the present application; Figure 6 Average body weight diagram of mice in Test Example 1 of the present application; Figure 7-1 Apoptosis effect diagram in Test Example 1 of the present application; Figure 7-2 Statistical diagram of apoptosis effect in Test Example 1 of the present application, wherein A is -NE group and B is +NE group; Figure 8-1 Cycle arrest statistical diagram in Test Example 1 of the present application, wherein A is -NE group and B is +NE group; Figure 8-2 Cycle arrest effect diagram in Test Example 1 of the present application; Figure 9 Cell level toxicity diagram in Test Example 1 of the present application. DETAILED DESCRIPTION

[0020] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is made for the purpose of illustrating the certain aspects, features and embodiments of the present application and is not intended to limit the scope of the application, which is defined solely by the appended claims.

[0021] It should be understood that the terms used herein are merely descriptive, but that the application should not be construed as being limited thereto. In addition, with respect to numerical ranges in the present application, it should be understood that each intermediate value between the upper and lower limits of the range is specifically disclosed. Each smaller range between any stated value or between any stated range is also included in the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The present invention provides a method for preparing an antibody conjugate Hu3F8-BMOA-MMAE, comprising the following steps: (1) Preparation of Fmoc-Bpa-Met(O2)-Oic-Abu-PAB: Fmoc-Bpa-Met(O2)-Oic-Abu was uniformly mixed with DCM, CH3OH, PABOH, and EEDQ, heated for reaction, dried, added with acetonitrile, and dried to obtain Fmoc-Bpa-Met(O2)-Oic-Abu-PAB; the mass volume ratio of Fmoc-Bpa-Met(O2)-Oic-Abu, DCM, CH3OH, PABOH, and EEDQ was 250 mg:5 mL:2.5 mL:140 mg:276 mg; the heating reaction temperature was 40°C, and the reaction time was 20-30 h.

[0026] (2) Preparation of Bpa-Met(O2)-Oic-Abu-PAB: Fmoc-Bpa-Met(O2)-Oic-Abu-PAB was mixed with DMF and DEA, reacted, and dried to obtain Bpa-Met(O 2) -Oic-Abu-PAB; the mass volume ratio of the Fmoc-Bpa-Met(O2)-Oic-Abu-PAB, DMF, and DEA is 200 mg:5 mL:0.625 mL; and the reaction time is 0.1~1 h.

[0027] (3) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB: Mal-PEG8-COOH, EDCI, and chloroform were evenly mixed, Bpa-Met(O2)-Oic-Abu-PAB was added, the mixture was reacted, dried, water and acetonitrile were added to dissolve the mixture, and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB; the mass volume ratio of Mal-PEG8-COOH, EDCI, chloroform, and Bpa-Met(O2)-Oic-Abu-PAB was 109 mg:35 mg:10 mL:95 mg; and the reaction time was 1 to 4 h.

[0028] (4) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP: Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB was uniformly mixed with DMF, DIEA and DNPC, reacted and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP; the mass volume ratio of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB to DMF, DIEA and DNPC was 120 mg:5 mL:23 mg:81 mg; and the reaction time was 8 to 16 h.

[0029] (5) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE: Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP was uniformly mixed with DMF, DIEA, HOBT and MMAE, reacted and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE; the mass volume ratio of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP to DMF, DIEA, HOBT and MMAE was 40 mg:3 mL:20 mg:7 mg:38 mg; and the reaction time was 4 to 16 h.

[0030] (6) The antibody Hu3F8 is replaced into buffer, TCEP is added, the reaction is stirred, Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE is added after cooling, the reaction is mixed, and an antibody conjugate Hu3F8-BMOA-MMAE is obtained.

[0031] Example 1

[0032] The embodiment 1 of the present application provides a preparation method of an antibody conjugate Hu3F8-BMOA-MMAE, and a flow chart is as shown in Figure 1 The specific steps are as follows: 1. Fmoc-Bpa-Met(O2)-Oic-Abu (250 mg, 1 eq, compound 1) is added into a 100 mL three-necked flask, DCM (5 mL) and CH3OH (2.5 mL) are added, then PABOH (70 mg, 2 eq) and EEDQ (138 mg, 2 eq) are sequentially added, a water bath is used for 40°C reaction for 24 h. HPLC inspection shows that 50% of the raw material remains, PABOH (2 eq) and EEDQ (2 eq) are added, and stirring is continued for 4 h, and the reaction is basically complete. After treatment, acetonitrile is added, and a conventional method is used for medium-pressure reverse phase, a mobile phase is selected to be 0.025% TFA water / acetonitrile, and freeze-drying is performed, 200 mg, yellow solid Fmoc-Bpa-Met(O2)-Oic-Abu-PAB (compound 2).

[0033] 2. Fmoc-Bpa-Met(O2)-Oic-Abu-PAB (200 mg) is added into a 50 mL single-necked flask, DMF (5 mL) is added, DEA (0.625 mL) is added at 0°C, and after dropwise addition is completed, room temperature reaction is performed for 0.5 h. HPLC detection shows that the reaction is complete. After treatment, a conventional method is used for medium-pressure reverse phase, a mobile phase is selected to be pure water / acetonitrile, and freeze-drying is performed, 130 mg, white solid Bpa-Met(O2)-Oic-Abu-PAB (compound 3).

[0034] 3. Mal-PEG8-COOH (109 mg, 1.5 eq), EDCI (35 mg, 1.5 eq) were added to a 50 mL single-neck flask, chloroform (10 mL) was added, stirred for 10 min, Bpa-Met(O2)-Oic-Abu-PAB (95 mg, 1 eq) was added and stirring was continued for 2 h. HPLC showed that the reaction was complete. After treatment, the reaction solution was rotary evaporated, water and acetonitrile were added to dissolve, and the product was obtained by adopting the conventional method of medium pressure reverse phase with pure water / acetonitrile=60% / 40%, and freeze-drying to obtain 144 mg of white solid Mal-amido-PEG8-Bpa-Met(O2)-Oic-Abu-PAB (compound 4).

[0035] 4. Mal-PEG8-Bpa-Met(O2)-Oic-Abu-PAB (120 mg, 1.0 eq) was added to a 50 mL single-neck flask, DMF (5 mL) was added, DIEA (23 mg, 2.0 eq) was added, and DNPC (81 mg, 3 eq) was stirred at 30°C overnight. HPLC showed that the reaction was complete. After treatment, the product was obtained by adopting the conventional method of medium pressure reverse phase with pure water / acetonitrile=55% / 45%, and freeze-drying to obtain 120 mg of white solid Mal-amido-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP (compound 5).

[0036] 5. Mal-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP (40 mg, 1.0 eq) was added to a 50 mL single-neck flask, DMF (3 mL) was added, DIEA (10 mg, 3.0 eq) was added, HOBT (7 mg, 2 eq) was added, and MMAE (38 mg, 2 eq) was stirred for 4 h. HPLC showed that half of the raw material and product were detected, DIEA (10 mg, 3.0 eq) was added, and a small amount of Mal-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP remained after 8 h. After treatment, high pressure preparation was adopted, and the product (MAP-BMOA-MMAE, compound 6) Mal-amido-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE was obtained by adopting the conventional method of medium pressure reverse phase with 0.05% TFA water / acetonitrile=35% / 65%, and freeze-drying to obtain 20 mg, HPLC: 92.47%.

[0037] 6. Replace the antibody XM001AB01 (Hu3F8) into the buffer required for the experiment (5 mM PB, 5 mM EDTA, pH 7.2), take 33.7 mg of the antibody (6.48 mg / mL, 5.2 mL), add 65.5 μL of TCEP (10 mM, 2.8 eq), stir at room temperature for 2 hours, cool to 0-5°C, add 375 μL of MAP-BMOA-MMAE (5 mM, DMSO, 8 eq), mix well, stir at room temperature for 30 minutes, and detect by HIC-HPLC. After the coupling is completed, the reaction solution is filtered with 20 mM His buffer (pH 6.0) to remove excess small molecules and organic solvents to obtain the antibody conjugate Hu3F8-BMOA-MMAE ( Figure 2 ), Hu3F8-BMOA-MMAE was tested by SEC-HPLC, and the results are shown in Figure 3 .

[0038] Figure 3 The DARS of Hu3F8-BMOA-MMAE calculated by HIC-HPLC test is about 4, which is in line with the expected coupling value range. Figure 4 SEC-HPLC analysis showed that Hu3F8-BMOA-MMAE accounted for 79.8% of the total product.

[0039] Test Example 1

[0040] Test Example 1 of the present invention detected the anti-tumor activity of the antibody conjugate Hu3F8-BMOA-MMAE prepared in Example 1 against GD2-positive tumor-bearing mice. The specific steps are as follows: M21 cell suspension (5 × 10 6 / mL / mouse) was inoculated into the right armpit of nude mice. When the tumor volume reached 80mm 3 The mice were divided into two groups, each with 6 mice. The treatment group was injected with 10 mg / kg body weight of the drug into the tail vein, and the control group was given the same amount of normal saline. The drug was administered once a week for four weeks. The body weight and tumor volume of the nude mice were recorded every 3-4 days during the period. After the fourth administration, the mice were observed for one week, sacrificed and photographed. The results are shown in the figure below. Figure 5 、 6 shown.

[0041] Figure 5 、 6 It was shown that compared with the control group, the antibody-coupled Hu3F8-BMOA-MMAE can significantly reduce tumor volume, has good tumor cell killing ability, and is highly safe.

[0042] Test Example 2

[0043] The apoptosis and cycle arrest of M21 cells by the Hu3F8-BMOA-MMAE prepared in Example 1 were detected in Test Example 2, and the specific steps are as follows: M21 cells were inoculated into 6-well cell culture plates at a density of 2 mL per well and 5.0x10 5 cells per well, and incubated overnight. The next day, Hu3F8-BMOA-MMAE was added to the cells at the following concentration gradients: 0, 1, 10, and 100 μg / mL, and enzyme-added and non-enzyme-added control groups were set up. After 48 h of drug uptake, the cells were collected and stained, and the data was detected and counted on a machine. The results are shown in Figure 7-1 、 Figure 7-2 、 Figure 8-1 、 Figure 8-2 .

[0044] Figure 7-1 、 Figure 7-2 、 Figure 8-1 、 Figure 8-2 It can be seen that with the increase of the dose, the apoptosis and cycle arrest of the cells also increase, which is dose-dependent. At the same time, the cycle and cell arrest of the enzyme-added experimental group are slightly increased compared with the non-enzyme-added experimental group.

[0045] Test Example 3

[0046] The toxicity of the Hu3F8-BMOA-MMAE prepared in Example 1 to GD2-positive M21 cells was detected in Test Example 3.

[0047] The specific steps are as follows: M21 cells were inoculated into 96-well cell culture plates at a density of 200 μL per well and 5.0x10 3 cells per well, and incubated overnight. The next day, Hu3F8-BMOA-MMAE was added to the cells at the following concentration gradients: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μg / mL, and enzyme-added and non-enzyme-added experimental control groups were set up. After 48 h of drug uptake, 20 μL of 5 mg / mL MTT solution was added to each well in the dark, and the plates were incubated at 37°C for 4 h. After the incubation, the drug-containing medium was discarded, 150 μL of DMSO was added to each well, and the plates were shaken for 5 min. The absorbance of each well at 570 nm was measured, and the cytotoxicity was calculated. The results are shown in Figure 9 .

[0048] Figure 9 It can be seen that the enzyme-added group has higher cytotoxicity compared with the non-enzyme-added group.

[0049] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. An antibody conjugate Hu3F8-BMOA-MMAE, wherein the antibody conjugate Hu3F8-BMOA-MMAE uses the BMOA sequence as a linker and couples MMAE to the antibody via cysteine ​​coupling technology.

2. A method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 1, characterized in that: The steps include: (1) Preparation of Fmoc-Bpa-Met(O2)-Oic-Abu-PAB: Fmoc-Bpa-Met(O2)-Oic-Abu was mixed with DCM, CH3OH, PABOH, and EEDQ, heated for reaction, dried, added with acetonitrile, and dried to obtain Fmoc-Bpa-Met(O2)-Oic-Abu-PAB; (2) Preparation of Bpa-Met(O2)-Oic-Abu-PAB: Fmoc-Bpa-Met(O2)-Oic-Abu-PAB was mixed with DMF and DEA, reacted, and dried to obtain Bpa-Met(O 2) -Oic-Abu-PAB; (3) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB: Mal-PEG8-COOH, EDCI, and chloroform were mixed evenly, and Bpa-Met(O2)-Oic-Abu-PAB was added, reacted, dried, dissolved in water and acetonitrile, and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB; (4) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP: Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB was mixed with DMF, DIEA and DNPC, reacted and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP; (5) Preparation of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE: Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP was mixed with DMF, DIEA, HOBT and MMAE, reacted and dried to obtain Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE; (6) Replace the antibody Hu3F8 into the buffer solution, add TCEP, stir the reaction, cool it down, add Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-MMAE, mix and react to obtain the antibody conjugate Hu3F8-BMOA-MMAE.

3. The method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 2, characterized in that: In step (1), the mass volume ratio of Fmoc-Bpa-Met(O2)-Oic-Abu, DCM, CH3OH, PABOH, and EEDQ is 200-300 mg:1-10 mL:1-5 mL:100-160 mg:200-300 mg; the temperature of the heating reaction is 40°C, and the time is 20-30 h.

4. The method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 2, wherein: In step (2), the mass volume ratio of the Fmoc-Bpa-Met(O2)-Oic-Abu-PAB, DMF, and DEA is 150-250 mg:2-8 mL:0.5-1.5 mL; and the reaction time is 0.1-1 h.

5. The method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 2, wherein: In step (3), the mass volume ratio of Mal-PEG8-COOH, EDCI, chloroform, and Bpa-Met(O2)-Oic-Abu-PAB is 90-120 mg:20-40 mg:5-15 mL:85-100 mg; and the reaction time is 1-4 h.

6. The method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 2, wherein: In step (4), the mass volume ratio of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB to DMF, DIEA, and DNPC is 100-150 mg:2-8 mL:2-30 mg:70-90 mg; and the reaction time is 8-16 h.

7. The method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 2, wherein: In step (5), the mass volume ratio of Mal-admio-PEG8-Bpa-Met(O2)-Oic-Abu-PAB-PNP to DMF, DIEA, HOBT, and MMAE is 30-50 mg:1-5 mL:10-30 mg:5-10 mg:30-40 mg; and the reaction time is 4-16 h.

8. The method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 2, wherein: In step (6), the buffer solution includes 5 mM PB and 5 mM EDTA; the mass volume ratio of the antibody to TCEP and MAP-BMOA-MMAE is 30-40 mg:60-70 µL:300-400 µL; the concentration of the antibody in the buffer solution is 6-7 mg / mL, the concentration of TCEP is 5-15 mM, and the concentration of MAP-BMOA-MMAE is 3-7 mM.

9. The method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to claim 2, characterized in that: In step (6), the stirring reaction time is 1 to 4 h, the cooling temperature is 0-5°C, and the mixing reaction time is 10 to 40 min.

10. Use of the antibody conjugate prepared by the method for preparing the antibody conjugate Hu3F8-BMOA-MMAE according to any one of claims 2 to 9 in preparing a drug for treating cancer.

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